work/1_panel_design/20180918_degenerate_primers.ipynb

{
 "cells": [
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "Create degenerate primer sequences from alignments and internal primer coordinates"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 32,
   "metadata": {},
   "outputs": [],
   "source": [
    "import re\n",
    "import sys\n",
    "import pandas as pd\n",
    "import numpy as np\n",
    "from Bio import AlignIO\n",
    "from Bio.Seq import Seq"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 2,
   "metadata": {},
   "outputs": [],
   "source": [
    "# in\n",
    "AMPL_FILE = \"data/20180706_comb_ampl.maf\"\n",
    "PRIMER_FILE = \"data/20180914_Simpledesigninfo_updated.xlsx\"\n",
    "# out\n",
    "OUT_FILE = \"data/20180918_Simpledesigninfo_updated.xlsx\""
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 6,
   "metadata": {},
   "outputs": [
    {
     "data": {
      "text/html": [
       "<div>\n",
       "<style scoped>\n",
       "    .dataframe tbody tr th:only-of-type {\n",
       "        vertical-align: middle;\n",
       "    }\n",
       "\n",
       "    .dataframe tbody tr th {\n",
       "        vertical-align: top;\n",
       "    }\n",
       "\n",
       "    .dataframe thead th {\n",
       "        text-align: right;\n",
       "    }\n",
       "</style>\n",
       "<table border=\"1\" class=\"dataframe\">\n",
       "  <thead>\n",
       "    <tr style=\"text-align: right;\">\n",
       "      <th></th>\n",
       "      <th>ID</th>\n",
       "      <th>TemplateID</th>\n",
       "      <th>ForwardPrimer(Fp)</th>\n",
       "      <th>ReversePrimer(Rp)</th>\n",
       "      <th>FpTm(Celsius degree)</th>\n",
       "      <th>RpTm(Celsius degree)</th>\n",
       "      <th>FpGC(%)</th>\n",
       "      <th>RpGC(%)</th>\n",
       "      <th>FpPos</th>\n",
       "      <th>RpPos</th>\n",
       "      <th>Penalty</th>\n",
       "      <th>AmpSize(bp)</th>\n",
       "      <th>SNPs in primer?</th>\n",
       "    </tr>\n",
       "  </thead>\n",
       "  <tbody>\n",
       "    <tr>\n",
       "      <th>0</th>\n",
       "      <td>1</td>\n",
       "      <td>531_cons</td>\n",
       "      <td>acattagtgctttattacgtggt</td>\n",
       "      <td>catttctttggcactttctgat</td>\n",
       "      <td>56.87</td>\n",
       "      <td>56.10</td>\n",
       "      <td>34.78</td>\n",
       "      <td>36.36</td>\n",
       "      <td>18</td>\n",
       "      <td>207</td>\n",
       "      <td>1.90</td>\n",
       "      <td>190</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>1</th>\n",
       "      <td>2</td>\n",
       "      <td>432_cons</td>\n",
       "      <td>acctgcattcgtatgtgttcta</td>\n",
       "      <td>ttcgaggtgggagattttcttc</td>\n",
       "      <td>58.12</td>\n",
       "      <td>58.65</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>5</td>\n",
       "      <td>217</td>\n",
       "      <td>0.65</td>\n",
       "      <td>213</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>2</th>\n",
       "      <td>3</td>\n",
       "      <td>473_var</td>\n",
       "      <td>tgctatggtacctaaacctacg</td>\n",
       "      <td>cgatgggtcaaacggaatcg</td>\n",
       "      <td>57.66</td>\n",
       "      <td>59.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>55.00</td>\n",
       "      <td>46</td>\n",
       "      <td>269</td>\n",
       "      <td>3.91</td>\n",
       "      <td>224</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>3</th>\n",
       "      <td>4</td>\n",
       "      <td>193_var</td>\n",
       "      <td>gccatgacgatatctacaatgc</td>\n",
       "      <td>gcgaacgctgattctaattttg</td>\n",
       "      <td>57.98</td>\n",
       "      <td>57.58</td>\n",
       "      <td>45.46</td>\n",
       "      <td>40.91</td>\n",
       "      <td>16</td>\n",
       "      <td>249</td>\n",
       "      <td>0.42</td>\n",
       "      <td>234</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>4</th>\n",
       "      <td>2</td>\n",
       "      <td>126_var</td>\n",
       "      <td>ttcaggagctcaccaaatcg</td>\n",
       "      <td>tccttcacctgttgtactgtat</td>\n",
       "      <td>58.48</td>\n",
       "      <td>57.08</td>\n",
       "      <td>50.00</td>\n",
       "      <td>40.91</td>\n",
       "      <td>123</td>\n",
       "      <td>314</td>\n",
       "      <td>0.92</td>\n",
       "      <td>192</td>\n",
       "      <td>low freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>5</th>\n",
       "      <td>3</td>\n",
       "      <td>416_var</td>\n",
       "      <td>aactggcactagtcatgtacat</td>\n",
       "      <td>caattaattccgttagcccgtg</td>\n",
       "      <td>57.77</td>\n",
       "      <td>58.63</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>19</td>\n",
       "      <td>212</td>\n",
       "      <td>0.63</td>\n",
       "      <td>194</td>\n",
       "      <td>low freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>6</th>\n",
       "      <td>4</td>\n",
       "      <td>495_var</td>\n",
       "      <td>ttcctcgcacactcatgtaag</td>\n",
       "      <td>tctcgatgtagaactgcacg</td>\n",
       "      <td>58.62</td>\n",
       "      <td>58.02</td>\n",
       "      <td>47.62</td>\n",
       "      <td>50.00</td>\n",
       "      <td>68</td>\n",
       "      <td>261</td>\n",
       "      <td>2.02</td>\n",
       "      <td>194</td>\n",
       "      <td>low freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>7</th>\n",
       "      <td>5</td>\n",
       "      <td>435_var</td>\n",
       "      <td>agttctttggagagttcggaat</td>\n",
       "      <td>aagaaaagaagtgagaaagcga</td>\n",
       "      <td>57.97</td>\n",
       "      <td>56.82</td>\n",
       "      <td>40.91</td>\n",
       "      <td>36.36</td>\n",
       "      <td>48</td>\n",
       "      <td>242</td>\n",
       "      <td>1.18</td>\n",
       "      <td>195</td>\n",
       "      <td>low freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>8</th>\n",
       "      <td>6</td>\n",
       "      <td>283_var</td>\n",
       "      <td>cacacaacttttatgtttaatca</td>\n",
       "      <td>aaaataatggccactattccc</td>\n",
       "      <td>52.53</td>\n",
       "      <td>54.45</td>\n",
       "      <td>26.09</td>\n",
       "      <td>38.10</td>\n",
       "      <td>53</td>\n",
       "      <td>249</td>\n",
       "      <td>4.55</td>\n",
       "      <td>197</td>\n",
       "      <td>low freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>9</th>\n",
       "      <td>10</td>\n",
       "      <td>205_cons</td>\n",
       "      <td>agacacaaggccttgaagaag</td>\n",
       "      <td>acagcttcttcttgtcatcgta</td>\n",
       "      <td>58.73</td>\n",
       "      <td>58.07</td>\n",
       "      <td>47.62</td>\n",
       "      <td>40.91</td>\n",
       "      <td>41</td>\n",
       "      <td>275</td>\n",
       "      <td>0.07</td>\n",
       "      <td>235</td>\n",
       "      <td>low freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>10</th>\n",
       "      <td>8</td>\n",
       "      <td>579_X</td>\n",
       "      <td>cacctgacactcgatggg</td>\n",
       "      <td>catgcagccggctacttg</td>\n",
       "      <td>57.65</td>\n",
       "      <td>59.46</td>\n",
       "      <td>61.11</td>\n",
       "      <td>61.11</td>\n",
       "      <td>0</td>\n",
       "      <td>191</td>\n",
       "      <td>5.46</td>\n",
       "      <td>192</td>\n",
       "      <td>high freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>11</th>\n",
       "      <td>10</td>\n",
       "      <td>575_X</td>\n",
       "      <td>agactttcaggccatgatacc</td>\n",
       "      <td>tcagtgaacgttgacttggtaa</td>\n",
       "      <td>58.03</td>\n",
       "      <td>58.46</td>\n",
       "      <td>47.62</td>\n",
       "      <td>40.91</td>\n",
       "      <td>11</td>\n",
       "      <td>202</td>\n",
       "      <td>0.46</td>\n",
       "      <td>192</td>\n",
       "      <td>high freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>12</th>\n",
       "      <td>17</td>\n",
       "      <td>576_X</td>\n",
       "      <td>aggagatgcaagttactatcgc</td>\n",
       "      <td>cactatcgtagcccaggttc</td>\n",
       "      <td>58.60</td>\n",
       "      <td>58.06</td>\n",
       "      <td>45.46</td>\n",
       "      <td>55.00</td>\n",
       "      <td>24</td>\n",
       "      <td>221</td>\n",
       "      <td>2.06</td>\n",
       "      <td>198</td>\n",
       "      <td>high freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>13</th>\n",
       "      <td>18</td>\n",
       "      <td>582_X</td>\n",
       "      <td>gaggcgcaggacacgatc</td>\n",
       "      <td>ctcctggatcttctgcatctc</td>\n",
       "      <td>61.14</td>\n",
       "      <td>58.19</td>\n",
       "      <td>66.67</td>\n",
       "      <td>52.38</td>\n",
       "      <td>24</td>\n",
       "      <td>224</td>\n",
       "      <td>1.19</td>\n",
       "      <td>201</td>\n",
       "      <td>high freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>14</th>\n",
       "      <td>22</td>\n",
       "      <td>587_X</td>\n",
       "      <td>tttcagcagaaacatccacatg</td>\n",
       "      <td>gtttcgatgtacttggtggttt</td>\n",
       "      <td>58.08</td>\n",
       "      <td>57.76</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>6</td>\n",
       "      <td>208</td>\n",
       "      <td>0.24</td>\n",
       "      <td>203</td>\n",
       "      <td>high freq</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>15</th>\n",
       "      <td>1</td>\n",
       "      <td>474_other</td>\n",
       "      <td>tgaattatgattaatggtgtgcc</td>\n",
       "      <td>caagtgggtgtgacattcca</td>\n",
       "      <td>55.92</td>\n",
       "      <td>58.61</td>\n",
       "      <td>34.78</td>\n",
       "      <td>50.00</td>\n",
       "      <td>13</td>\n",
       "      <td>202</td>\n",
       "      <td>2.61</td>\n",
       "      <td>190</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>16</th>\n",
       "      <td>3</td>\n",
       "      <td>433_other</td>\n",
       "      <td>aatgcttccattaactgccatt</td>\n",
       "      <td>ggttaagttttaatgtgcgtaaca</td>\n",
       "      <td>57.50</td>\n",
       "      <td>57.05</td>\n",
       "      <td>36.36</td>\n",
       "      <td>33.33</td>\n",
       "      <td>23</td>\n",
       "      <td>212</td>\n",
       "      <td>2.95</td>\n",
       "      <td>190</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>17</th>\n",
       "      <td>4</td>\n",
       "      <td>298_other</td>\n",
       "      <td>tagaattacaatcaagtggaagacg</td>\n",
       "      <td>ttatacagtggatggcagtcg</td>\n",
       "      <td>57.76</td>\n",
       "      <td>58.13</td>\n",
       "      <td>36.00</td>\n",
       "      <td>47.62</td>\n",
       "      <td>52</td>\n",
       "      <td>241</td>\n",
       "      <td>1.13</td>\n",
       "      <td>190</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>18</th>\n",
       "      <td>5</td>\n",
       "      <td>356_other</td>\n",
       "      <td>tctatcaccatgcacttcttgt</td>\n",
       "      <td>aatgtagataggctaaaaccgc</td>\n",
       "      <td>58.04</td>\n",
       "      <td>56.89</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40</td>\n",
       "      <td>239</td>\n",
       "      <td>1.11</td>\n",
       "      <td>200</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>19</th>\n",
       "      <td>6</td>\n",
       "      <td>439_other</td>\n",
       "      <td>gcgcgtaatcgttttatcaaga</td>\n",
       "      <td>acaaatctgattacgatatgatc</td>\n",
       "      <td>58.39</td>\n",
       "      <td>52.88</td>\n",
       "      <td>40.91</td>\n",
       "      <td>30.44</td>\n",
       "      <td>47</td>\n",
       "      <td>247</td>\n",
       "      <td>6.12</td>\n",
       "      <td>201</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>20</th>\n",
       "      <td>7</td>\n",
       "      <td>269_other</td>\n",
       "      <td>aatgattcggatgatttgcttc</td>\n",
       "      <td>ccgtcattatttcccaatcaaa</td>\n",
       "      <td>56.06</td>\n",
       "      <td>55.63</td>\n",
       "      <td>36.36</td>\n",
       "      <td>36.36</td>\n",
       "      <td>12</td>\n",
       "      <td>214</td>\n",
       "      <td>2.37</td>\n",
       "      <td>203</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>21</th>\n",
       "      <td>9</td>\n",
       "      <td>140_other</td>\n",
       "      <td>gtgaattagattttaatgaagtaa</td>\n",
       "      <td>agtgggcagatttaattttagatct</td>\n",
       "      <td>50.09</td>\n",
       "      <td>57.16</td>\n",
       "      <td>20.83</td>\n",
       "      <td>32.00</td>\n",
       "      <td>2</td>\n",
       "      <td>217</td>\n",
       "      <td>3.84</td>\n",
       "      <td>216</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>22</th>\n",
       "      <td>10</td>\n",
       "      <td>245_other</td>\n",
       "      <td>cgcaattgtcacaaatctgtat</td>\n",
       "      <td>gcagtttacggcttttctcc</td>\n",
       "      <td>56.29</td>\n",
       "      <td>57.95</td>\n",
       "      <td>36.36</td>\n",
       "      <td>50.00</td>\n",
       "      <td>8</td>\n",
       "      <td>230</td>\n",
       "      <td>2.05</td>\n",
       "      <td>223</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>23</th>\n",
       "      <td>12</td>\n",
       "      <td>65_other</td>\n",
       "      <td>gcaaaatttccgtcccatta</td>\n",
       "      <td>tgtaattagctgtgtcttgtg</td>\n",
       "      <td>55.11</td>\n",
       "      <td>54.46</td>\n",
       "      <td>40.00</td>\n",
       "      <td>38.10</td>\n",
       "      <td>4</td>\n",
       "      <td>235</td>\n",
       "      <td>4.54</td>\n",
       "      <td>232</td>\n",
       "      <td>None</td>\n",
       "    </tr>\n",
       "  </tbody>\n",
       "</table>\n",
       "</div>"
      ],
      "text/plain": [
       "    ID TemplateID          ForwardPrimer(Fp)          ReversePrimer(Rp)  \\\n",
       "0    1   531_cons    acattagtgctttattacgtggt     catttctttggcactttctgat   \n",
       "1    2   432_cons     acctgcattcgtatgtgttcta     ttcgaggtgggagattttcttc   \n",
       "2    3    473_var     tgctatggtacctaaacctacg       cgatgggtcaaacggaatcg   \n",
       "3    4    193_var     gccatgacgatatctacaatgc     gcgaacgctgattctaattttg   \n",
       "4    2    126_var       ttcaggagctcaccaaatcg     tccttcacctgttgtactgtat   \n",
       "5    3    416_var     aactggcactagtcatgtacat     caattaattccgttagcccgtg   \n",
       "6    4    495_var      ttcctcgcacactcatgtaag       tctcgatgtagaactgcacg   \n",
       "7    5    435_var     agttctttggagagttcggaat     aagaaaagaagtgagaaagcga   \n",
       "8    6    283_var    cacacaacttttatgtttaatca      aaaataatggccactattccc   \n",
       "9   10   205_cons      agacacaaggccttgaagaag     acagcttcttcttgtcatcgta   \n",
       "10   8      579_X         cacctgacactcgatggg         catgcagccggctacttg   \n",
       "11  10      575_X      agactttcaggccatgatacc     tcagtgaacgttgacttggtaa   \n",
       "12  17      576_X     aggagatgcaagttactatcgc       cactatcgtagcccaggttc   \n",
       "13  18      582_X         gaggcgcaggacacgatc      ctcctggatcttctgcatctc   \n",
       "14  22      587_X     tttcagcagaaacatccacatg     gtttcgatgtacttggtggttt   \n",
       "15   1  474_other    tgaattatgattaatggtgtgcc       caagtgggtgtgacattcca   \n",
       "16   3  433_other     aatgcttccattaactgccatt   ggttaagttttaatgtgcgtaaca   \n",
       "17   4  298_other  tagaattacaatcaagtggaagacg      ttatacagtggatggcagtcg   \n",
       "18   5  356_other     tctatcaccatgcacttcttgt     aatgtagataggctaaaaccgc   \n",
       "19   6  439_other     gcgcgtaatcgttttatcaaga    acaaatctgattacgatatgatc   \n",
       "20   7  269_other     aatgattcggatgatttgcttc     ccgtcattatttcccaatcaaa   \n",
       "21   9  140_other   gtgaattagattttaatgaagtaa  agtgggcagatttaattttagatct   \n",
       "22  10  245_other     cgcaattgtcacaaatctgtat       gcagtttacggcttttctcc   \n",
       "23  12   65_other       gcaaaatttccgtcccatta      tgtaattagctgtgtcttgtg   \n",
       "\n",
       "    FpTm(Celsius degree)  RpTm(Celsius degree)  FpGC(%)  RpGC(%)  FpPos  \\\n",
       "0                  56.87                 56.10    34.78    36.36     18   \n",
       "1                  58.12                 58.65    40.91    45.46      5   \n",
       "2                  57.66                 59.91    45.46    55.00     46   \n",
       "3                  57.98                 57.58    45.46    40.91     16   \n",
       "4                  58.48                 57.08    50.00    40.91    123   \n",
       "5                  57.77                 58.63    40.91    45.46     19   \n",
       "6                  58.62                 58.02    47.62    50.00     68   \n",
       "7                  57.97                 56.82    40.91    36.36     48   \n",
       "8                  52.53                 54.45    26.09    38.10     53   \n",
       "9                  58.73                 58.07    47.62    40.91     41   \n",
       "10                 57.65                 59.46    61.11    61.11      0   \n",
       "11                 58.03                 58.46    47.62    40.91     11   \n",
       "12                 58.60                 58.06    45.46    55.00     24   \n",
       "13                 61.14                 58.19    66.67    52.38     24   \n",
       "14                 58.08                 57.76    40.91    40.91      6   \n",
       "15                 55.92                 58.61    34.78    50.00     13   \n",
       "16                 57.50                 57.05    36.36    33.33     23   \n",
       "17                 57.76                 58.13    36.00    47.62     52   \n",
       "18                 58.04                 56.89    40.91    40.91     40   \n",
       "19                 58.39                 52.88    40.91    30.44     47   \n",
       "20                 56.06                 55.63    36.36    36.36     12   \n",
       "21                 50.09                 57.16    20.83    32.00      2   \n",
       "22                 56.29                 57.95    36.36    50.00      8   \n",
       "23                 55.11                 54.46    40.00    38.10      4   \n",
       "\n",
       "    RpPos  Penalty  AmpSize(bp) SNPs in primer?  \n",
       "0     207     1.90          190            None  \n",
       "1     217     0.65          213            None  \n",
       "2     269     3.91          224            None  \n",
       "3     249     0.42          234            None  \n",
       "4     314     0.92          192        low freq  \n",
       "5     212     0.63          194        low freq  \n",
       "6     261     2.02          194        low freq  \n",
       "7     242     1.18          195        low freq  \n",
       "8     249     4.55          197        low freq  \n",
       "9     275     0.07          235        low freq  \n",
       "10    191     5.46          192       high freq  \n",
       "11    202     0.46          192       high freq  \n",
       "12    221     2.06          198       high freq  \n",
       "13    224     1.19          201       high freq  \n",
       "14    208     0.24          203       high freq  \n",
       "15    202     2.61          190            None  \n",
       "16    212     2.95          190            None  \n",
       "17    241     1.13          190            None  \n",
       "18    239     1.11          200            None  \n",
       "19    247     6.12          201            None  \n",
       "20    214     2.37          203            None  \n",
       "21    217     3.84          216            None  \n",
       "22    230     2.05          223            None  \n",
       "23    235     4.54          232            None  "
      ]
     },
     "execution_count": 6,
     "metadata": {},
     "output_type": "execute_result"
    }
   ],
   "source": [
    "alns = list(AlignIO.parse(AMPL_FILE, \"maf\"))\n",
    "primers = pd.read_excel(PRIMER_FILE, sheet_name=None)\n",
    "primers['Multiplex_core']"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 7,
   "metadata": {},
   "outputs": [
    {
     "data": {
      "text/plain": [
       "7"
      ]
     },
     "execution_count": 7,
     "metadata": {},
     "output_type": "execute_result"
    }
   ],
   "source": [
    "def gapped_coord(seq, coord):\n",
    "    '''\n",
    "    Coordinate of i-th letter in sequence within gapped sequence representation\n",
    "    '''\n",
    "    i = 0 # nucleotides\n",
    "    j = 0 # coordinates\n",
    "    while i < coord:\n",
    "        if str(seq[j]) != '-':\n",
    "            i += 1\n",
    "        j += 1\n",
    "    return j\n",
    "gapped_coord('-A-TA-C', 4)"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 65,
   "metadata": {},
   "outputs": [
    {
     "name": "stdout",
     "output_type": "stream",
     "text": [
      "CCTTCATgAACAGACGGTTGT\n",
      "GAGCACAT[C/T]AT[T/C]GCCGGT\n"
     ]
    }
   ],
   "source": [
    "def get_primer(alignment, alnid, primer, reverse=False, min_alts=2):\n",
    "    '''\n",
    "    Locate primer sequence in first sequence in the alignment, \n",
    "    return primer with ambiguities coded as [ref/alt/...].\n",
    "    \n",
    "    reverse - primer located on reverse strand\n",
    "    \n",
    "    min_alts - minimum number of genomes with alt alleles to be treated as ambiguous.\n",
    "    Positions with lower number of alts are converted to lowercase.\n",
    "    '''\n",
    "#     print(alnid)\n",
    "#     print(primer)\n",
    "#     print(alignment)\n",
    "    \n",
    "    seq = ''\n",
    "    alnid = int(alnid)\n",
    "    # search in 0-th sequence without gaps\n",
    "    source = str(alignment[alnid][0].seq).replace('-','')\n",
    "    \n",
    "    if reverse:\n",
    "        primer = str(Seq(primer).reverse_complement())\n",
    "    pmatch = re.search(primer, source, re.IGNORECASE)\n",
    "    # reverse strand primer\n",
    "    if reverse:\n",
    "        for i in range(gapped_coord(alignment[alnid][0], pmatch.end()) - 1,\n",
    "                       gapped_coord(alignment[alnid][0], pmatch.start()) - 1,\n",
    "                       -1):\n",
    "            col = [str(Seq(nt).reverse_complement()) for nt in alignment[alnid][:, i]]\n",
    "            ref = col[0]\n",
    "            alt = set(col)\n",
    "            alt.remove(ref)\n",
    "            if len(alt) == 0: # no alts\n",
    "                seq += ref\n",
    "            elif len(alt) == 1 and col.count(''.join(alt)) < min_alts: # single occurence of alt in alignment\n",
    "                seq += ref.lower()\n",
    "            else:\n",
    "                #print(''.join(col))\n",
    "                seq += '[{}/{}]'.format(ref, '/'.join(alt))\n",
    "    # forward strand primer\n",
    "    else:\n",
    "        for i in range(gapped_coord(alignment[alnid][0], pmatch.start()), gapped_coord(alignment[alnid][0], pmatch.end())):\n",
    "            col = alignment[alnid][:, i]\n",
    "            ref = col[0]\n",
    "            alt = set(col)\n",
    "            alt.remove(ref)\n",
    "            if len(set(col)) == 1:\n",
    "                seq += ref\n",
    "            elif len(alt) == 1 and col.count(''.join(alt)) < min_alts: # single occurence of alt in alignment\n",
    "                seq += ref.lower()\n",
    "            else:\n",
    "                alt = set(col)\n",
    "                alt.remove(ref)\n",
    "                seq += '[{}/{}]'.format(ref, '/'.join(alt))\n",
    "    \n",
    "    # reverse\n",
    "    \n",
    "        \n",
    "    return seq\n",
    "\n",
    "print(get_primer(alns, 508, 'ccttcatgaacagacggttgt'))\n",
    "print(get_primer(alns, 508, 'gagcacatcattgccggt', reverse=True))"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 67,
   "metadata": {},
   "outputs": [
    {
     "data": {
      "text/html": [
       "<div>\n",
       "<style scoped>\n",
       "    .dataframe tbody tr th:only-of-type {\n",
       "        vertical-align: middle;\n",
       "    }\n",
       "\n",
       "    .dataframe tbody tr th {\n",
       "        vertical-align: top;\n",
       "    }\n",
       "\n",
       "    .dataframe thead th {\n",
       "        text-align: right;\n",
       "    }\n",
       "</style>\n",
       "<table border=\"1\" class=\"dataframe\">\n",
       "  <thead>\n",
       "    <tr style=\"text-align: right;\">\n",
       "      <th></th>\n",
       "      <th>ID</th>\n",
       "      <th>TemplateID</th>\n",
       "      <th>ForwardPrimer(Fp)</th>\n",
       "      <th>ReversePrimer(Rp)</th>\n",
       "      <th>FpTm(Celsius degree)</th>\n",
       "      <th>RpTm(Celsius degree)</th>\n",
       "      <th>FpGC(%)</th>\n",
       "      <th>RpGC(%)</th>\n",
       "      <th>FpPos</th>\n",
       "      <th>RpPos</th>\n",
       "      <th>Penalty</th>\n",
       "      <th>AmpSize(bp)</th>\n",
       "      <th>SNPs in primer?</th>\n",
       "      <th>AlnID</th>\n",
       "      <th>fp_deg</th>\n",
       "      <th>rp_deg</th>\n",
       "    </tr>\n",
       "  </thead>\n",
       "  <tbody>\n",
       "    <tr>\n",
       "      <th>0</th>\n",
       "      <td>1</td>\n",
       "      <td>531_cons</td>\n",
       "      <td>acattagtgctttattacgtggt</td>\n",
       "      <td>catttctttggcactttctgat</td>\n",
       "      <td>56.87</td>\n",
       "      <td>56.10</td>\n",
       "      <td>34.78</td>\n",
       "      <td>36.36</td>\n",
       "      <td>18</td>\n",
       "      <td>207</td>\n",
       "      <td>1.90</td>\n",
       "      <td>190</td>\n",
       "      <td>None</td>\n",
       "      <td>531</td>\n",
       "      <td>ACATTAGTGCTTTATTACGTGGT</td>\n",
       "      <td>CATTTCTTTGGCACTTTCTGAT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>1</th>\n",
       "      <td>2</td>\n",
       "      <td>432_cons</td>\n",
       "      <td>acctgcattcgtatgtgttcta</td>\n",
       "      <td>ttcgaggtgggagattttcttc</td>\n",
       "      <td>58.12</td>\n",
       "      <td>58.65</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>5</td>\n",
       "      <td>217</td>\n",
       "      <td>0.65</td>\n",
       "      <td>213</td>\n",
       "      <td>None</td>\n",
       "      <td>432</td>\n",
       "      <td>ACCTGCATTCGTATGTGTTCTA</td>\n",
       "      <td>TTCGAGGTGGGAGATTTTCTTC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>2</th>\n",
       "      <td>3</td>\n",
       "      <td>473_var</td>\n",
       "      <td>tgctatggtacctaaacctacg</td>\n",
       "      <td>cgatgggtcaaacggaatcg</td>\n",
       "      <td>57.66</td>\n",
       "      <td>59.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>55.00</td>\n",
       "      <td>46</td>\n",
       "      <td>269</td>\n",
       "      <td>3.91</td>\n",
       "      <td>224</td>\n",
       "      <td>None</td>\n",
       "      <td>473</td>\n",
       "      <td>TGCTATGGTACCTAAACCTACG</td>\n",
       "      <td>CGATGGGTCAAACGGAATCG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>3</th>\n",
       "      <td>4</td>\n",
       "      <td>193_var</td>\n",
       "      <td>gccatgacgatatctacaatgc</td>\n",
       "      <td>gcgaacgctgattctaattttg</td>\n",
       "      <td>57.98</td>\n",
       "      <td>57.58</td>\n",
       "      <td>45.46</td>\n",
       "      <td>40.91</td>\n",
       "      <td>16</td>\n",
       "      <td>249</td>\n",
       "      <td>0.42</td>\n",
       "      <td>234</td>\n",
       "      <td>None</td>\n",
       "      <td>193</td>\n",
       "      <td>GCCATGACGATATCTACAATGC</td>\n",
       "      <td>GCGAACGCTGATTCTAATTTTG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>4</th>\n",
       "      <td>2</td>\n",
       "      <td>126_var</td>\n",
       "      <td>ttcaggagctcaccaaatcg</td>\n",
       "      <td>tccttcacctgttgtactgtat</td>\n",
       "      <td>58.48</td>\n",
       "      <td>57.08</td>\n",
       "      <td>50.00</td>\n",
       "      <td>40.91</td>\n",
       "      <td>123</td>\n",
       "      <td>314</td>\n",
       "      <td>0.92</td>\n",
       "      <td>192</td>\n",
       "      <td>low freq</td>\n",
       "      <td>126</td>\n",
       "      <td>TTCAGGAGCTCACCAAATCG</td>\n",
       "      <td>TCCTTCACCTGTTGTACTGTAT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>5</th>\n",
       "      <td>3</td>\n",
       "      <td>416_var</td>\n",
       "      <td>aactggcactagtcatgtacat</td>\n",
       "      <td>caattaattccgttagcccgtg</td>\n",
       "      <td>57.77</td>\n",
       "      <td>58.63</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>19</td>\n",
       "      <td>212</td>\n",
       "      <td>0.63</td>\n",
       "      <td>194</td>\n",
       "      <td>low freq</td>\n",
       "      <td>416</td>\n",
       "      <td>AACTgGCACTAGTCATGTACAT</td>\n",
       "      <td>CAATTAATTCCGTTAGCCCGTG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>6</th>\n",
       "      <td>4</td>\n",
       "      <td>495_var</td>\n",
       "      <td>ttcctcgcacactcatgtaag</td>\n",
       "      <td>tctcgatgtagaactgcacg</td>\n",
       "      <td>58.62</td>\n",
       "      <td>58.02</td>\n",
       "      <td>47.62</td>\n",
       "      <td>50.00</td>\n",
       "      <td>68</td>\n",
       "      <td>261</td>\n",
       "      <td>2.02</td>\n",
       "      <td>194</td>\n",
       "      <td>low freq</td>\n",
       "      <td>495</td>\n",
       "      <td>TTCCTCGCACACTCATGTAAG</td>\n",
       "      <td>TCTCGATGTAGAACTGCACg</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>7</th>\n",
       "      <td>5</td>\n",
       "      <td>435_var</td>\n",
       "      <td>agttctttggagagttcggaat</td>\n",
       "      <td>aagaaaagaagtgagaaagcga</td>\n",
       "      <td>57.97</td>\n",
       "      <td>56.82</td>\n",
       "      <td>40.91</td>\n",
       "      <td>36.36</td>\n",
       "      <td>48</td>\n",
       "      <td>242</td>\n",
       "      <td>1.18</td>\n",
       "      <td>195</td>\n",
       "      <td>low freq</td>\n",
       "      <td>435</td>\n",
       "      <td>AGTTCTTTGGAGAGTTCgGAAT</td>\n",
       "      <td>AAGAAAAGAAGTGAGAAAGCGA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>8</th>\n",
       "      <td>6</td>\n",
       "      <td>283_var</td>\n",
       "      <td>cacacaacttttatgtttaatca</td>\n",
       "      <td>aaaataatggccactattccc</td>\n",
       "      <td>52.53</td>\n",
       "      <td>54.45</td>\n",
       "      <td>26.09</td>\n",
       "      <td>38.10</td>\n",
       "      <td>53</td>\n",
       "      <td>249</td>\n",
       "      <td>4.55</td>\n",
       "      <td>197</td>\n",
       "      <td>low freq</td>\n",
       "      <td>283</td>\n",
       "      <td>CAC-ACAACTTTTATGTTTAATCA</td>\n",
       "      <td>AAAaTAATGGCCACTATTCCC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>9</th>\n",
       "      <td>10</td>\n",
       "      <td>205_cons</td>\n",
       "      <td>agacacaaggccttgaagaag</td>\n",
       "      <td>acagcttcttcttgtcatcgta</td>\n",
       "      <td>58.73</td>\n",
       "      <td>58.07</td>\n",
       "      <td>47.62</td>\n",
       "      <td>40.91</td>\n",
       "      <td>41</td>\n",
       "      <td>275</td>\n",
       "      <td>0.07</td>\n",
       "      <td>235</td>\n",
       "      <td>low freq</td>\n",
       "      <td>205</td>\n",
       "      <td>AGACACAAGGCCTTGAAGAAG</td>\n",
       "      <td>ACAGCTTCTTCTTGTCaTCGTA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>10</th>\n",
       "      <td>8</td>\n",
       "      <td>579_X</td>\n",
       "      <td>cacctgacactcgatggg</td>\n",
       "      <td>catgcagccggctacttg</td>\n",
       "      <td>57.65</td>\n",
       "      <td>59.46</td>\n",
       "      <td>61.11</td>\n",
       "      <td>61.11</td>\n",
       "      <td>0</td>\n",
       "      <td>191</td>\n",
       "      <td>5.46</td>\n",
       "      <td>192</td>\n",
       "      <td>high freq</td>\n",
       "      <td>579</td>\n",
       "      <td>[C/T]ACCTGACA[C/T]TCGAT[G/C]GG</td>\n",
       "      <td>[C/-][A/C/-][T/-/C][G/-/C][C/-/T]AGCCGGCTA[C/T...</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>11</th>\n",
       "      <td>10</td>\n",
       "      <td>575_X</td>\n",
       "      <td>agactttcaggccatgatacc</td>\n",
       "      <td>tcagtgaacgttgacttggtaa</td>\n",
       "      <td>58.03</td>\n",
       "      <td>58.46</td>\n",
       "      <td>47.62</td>\n",
       "      <td>40.91</td>\n",
       "      <td>11</td>\n",
       "      <td>202</td>\n",
       "      <td>0.46</td>\n",
       "      <td>192</td>\n",
       "      <td>high freq</td>\n",
       "      <td>575</td>\n",
       "      <td>aGA[C/T]TTTCAGGCCATGAT[A/C]CC</td>\n",
       "      <td>TC[A/C/T/G]GTGAA[C/G]GTTGA[C/T]TTgGTAA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>12</th>\n",
       "      <td>17</td>\n",
       "      <td>576_X</td>\n",
       "      <td>aggagatgcaagttactatcgc</td>\n",
       "      <td>cactatcgtagcccaggttc</td>\n",
       "      <td>58.60</td>\n",
       "      <td>58.06</td>\n",
       "      <td>45.46</td>\n",
       "      <td>55.00</td>\n",
       "      <td>24</td>\n",
       "      <td>221</td>\n",
       "      <td>2.06</td>\n",
       "      <td>198</td>\n",
       "      <td>high freq</td>\n",
       "      <td>576</td>\n",
       "      <td>AGGA[G/A]ATGCAAGTTACTATcGC</td>\n",
       "      <td>C[A/G]CTATCGTAGCCCAG[G/A]TTC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>13</th>\n",
       "      <td>18</td>\n",
       "      <td>582_X</td>\n",
       "      <td>gaggcgcaggacacgatc</td>\n",
       "      <td>ctcctggatcttctgcatctc</td>\n",
       "      <td>61.14</td>\n",
       "      <td>58.19</td>\n",
       "      <td>66.67</td>\n",
       "      <td>52.38</td>\n",
       "      <td>24</td>\n",
       "      <td>224</td>\n",
       "      <td>1.19</td>\n",
       "      <td>201</td>\n",
       "      <td>high freq</td>\n",
       "      <td>582</td>\n",
       "      <td>GAGGC[G/A/T]CAGGACACgATc</td>\n",
       "      <td>cTCCTGGATCTTCTGCATCTC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>14</th>\n",
       "      <td>22</td>\n",
       "      <td>587_X</td>\n",
       "      <td>tttcagcagaaacatccacatg</td>\n",
       "      <td>gtttcgatgtacttggtggttt</td>\n",
       "      <td>58.08</td>\n",
       "      <td>57.76</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>6</td>\n",
       "      <td>208</td>\n",
       "      <td>0.24</td>\n",
       "      <td>203</td>\n",
       "      <td>high freq</td>\n",
       "      <td>587</td>\n",
       "      <td>TTtCAGCAGAAACATCCACATG</td>\n",
       "      <td>GT[T/C]TCGATGTACTTGGTGGTTT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>15</th>\n",
       "      <td>1</td>\n",
       "      <td>474_other</td>\n",
       "      <td>tgaattatgattaatggtgtgcc</td>\n",
       "      <td>caagtgggtgtgacattcca</td>\n",
       "      <td>55.92</td>\n",
       "      <td>58.61</td>\n",
       "      <td>34.78</td>\n",
       "      <td>50.00</td>\n",
       "      <td>13</td>\n",
       "      <td>202</td>\n",
       "      <td>2.61</td>\n",
       "      <td>190</td>\n",
       "      <td>None</td>\n",
       "      <td>474</td>\n",
       "      <td>TGAATTATGATTAATGGTGTGCC</td>\n",
       "      <td>CAAGTGGGTGTGACATTCCA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>16</th>\n",
       "      <td>3</td>\n",
       "      <td>433_other</td>\n",
       "      <td>aatgcttccattaactgccatt</td>\n",
       "      <td>ggttaagttttaatgtgcgtaaca</td>\n",
       "      <td>57.50</td>\n",
       "      <td>57.05</td>\n",
       "      <td>36.36</td>\n",
       "      <td>33.33</td>\n",
       "      <td>23</td>\n",
       "      <td>212</td>\n",
       "      <td>2.95</td>\n",
       "      <td>190</td>\n",
       "      <td>None</td>\n",
       "      <td>433</td>\n",
       "      <td>AATGCTTCCATTAACTGCCATT</td>\n",
       "      <td>GGTTAAGTTTTAATGTGCGTAACA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>17</th>\n",
       "      <td>4</td>\n",
       "      <td>298_other</td>\n",
       "      <td>tagaattacaatcaagtggaagacg</td>\n",
       "      <td>ttatacagtggatggcagtcg</td>\n",
       "      <td>57.76</td>\n",
       "      <td>58.13</td>\n",
       "      <td>36.00</td>\n",
       "      <td>47.62</td>\n",
       "      <td>52</td>\n",
       "      <td>241</td>\n",
       "      <td>1.13</td>\n",
       "      <td>190</td>\n",
       "      <td>None</td>\n",
       "      <td>298</td>\n",
       "      <td>TAGAATTACAATCAAGTGGAAGACG</td>\n",
       "      <td>TTATACAGTGGATGGCAGTCG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>18</th>\n",
       "      <td>5</td>\n",
       "      <td>356_other</td>\n",
       "      <td>tctatcaccatgcacttcttgt</td>\n",
       "      <td>aatgtagataggctaaaaccgc</td>\n",
       "      <td>58.04</td>\n",
       "      <td>56.89</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40</td>\n",
       "      <td>239</td>\n",
       "      <td>1.11</td>\n",
       "      <td>200</td>\n",
       "      <td>None</td>\n",
       "      <td>356</td>\n",
       "      <td>TCTATCACCATGCACTTCTTGT</td>\n",
       "      <td>AATGTAGATAGGCTAAAACCGC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>19</th>\n",
       "      <td>6</td>\n",
       "      <td>439_other</td>\n",
       "      <td>gcgcgtaatcgttttatcaaga</td>\n",
       "      <td>acaaatctgattacgatatgatc</td>\n",
       "      <td>58.39</td>\n",
       "      <td>52.88</td>\n",
       "      <td>40.91</td>\n",
       "      <td>30.44</td>\n",
       "      <td>47</td>\n",
       "      <td>247</td>\n",
       "      <td>6.12</td>\n",
       "      <td>201</td>\n",
       "      <td>None</td>\n",
       "      <td>439</td>\n",
       "      <td>GCGCGTAATCGTTTTATCAAGA</td>\n",
       "      <td>ACAAATCTGATTACGATATGATC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>20</th>\n",
       "      <td>7</td>\n",
       "      <td>269_other</td>\n",
       "      <td>aatgattcggatgatttgcttc</td>\n",
       "      <td>ccgtcattatttcccaatcaaa</td>\n",
       "      <td>56.06</td>\n",
       "      <td>55.63</td>\n",
       "      <td>36.36</td>\n",
       "      <td>36.36</td>\n",
       "      <td>12</td>\n",
       "      <td>214</td>\n",
       "      <td>2.37</td>\n",
       "      <td>203</td>\n",
       "      <td>None</td>\n",
       "      <td>269</td>\n",
       "      <td>AATGATTCGGATGATTTGCTTC</td>\n",
       "      <td>CCGTCATTATTTCCCAATCAAA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>21</th>\n",
       "      <td>9</td>\n",
       "      <td>140_other</td>\n",
       "      <td>gtgaattagattttaatgaagtaa</td>\n",
       "      <td>agtgggcagatttaattttagatct</td>\n",
       "      <td>50.09</td>\n",
       "      <td>57.16</td>\n",
       "      <td>20.83</td>\n",
       "      <td>32.00</td>\n",
       "      <td>2</td>\n",
       "      <td>217</td>\n",
       "      <td>3.84</td>\n",
       "      <td>216</td>\n",
       "      <td>None</td>\n",
       "      <td>140</td>\n",
       "      <td>GTGAATTAGATTTTAATGAAGTAA</td>\n",
       "      <td>AGTGGGCAGATTTAATTTTAGATCT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>22</th>\n",
       "      <td>10</td>\n",
       "      <td>245_other</td>\n",
       "      <td>cgcaattgtcacaaatctgtat</td>\n",
       "      <td>gcagtttacggcttttctcc</td>\n",
       "      <td>56.29</td>\n",
       "      <td>57.95</td>\n",
       "      <td>36.36</td>\n",
       "      <td>50.00</td>\n",
       "      <td>8</td>\n",
       "      <td>230</td>\n",
       "      <td>2.05</td>\n",
       "      <td>223</td>\n",
       "      <td>None</td>\n",
       "      <td>245</td>\n",
       "      <td>---CGCAATTGTCACAAATCTGTAT</td>\n",
       "      <td>GCAGTTTACGGCTTTTCTCC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>23</th>\n",
       "      <td>12</td>\n",
       "      <td>65_other</td>\n",
       "      <td>gcaaaatttccgtcccatta</td>\n",
       "      <td>tgtaattagctgtgtcttgtg</td>\n",
       "      <td>55.11</td>\n",
       "      <td>54.46</td>\n",
       "      <td>40.00</td>\n",
       "      <td>38.10</td>\n",
       "      <td>4</td>\n",
       "      <td>235</td>\n",
       "      <td>4.54</td>\n",
       "      <td>232</td>\n",
       "      <td>None</td>\n",
       "      <td>65</td>\n",
       "      <td>GCAAAATTTCCGTCCCATTA</td>\n",
       "      <td>TGTAATTAGCTGTGTCTTGTG</td>\n",
       "    </tr>\n",
       "  </tbody>\n",
       "</table>\n",
       "</div>"
      ],
      "text/plain": [
       "    ID TemplateID          ForwardPrimer(Fp)          ReversePrimer(Rp)  \\\n",
       "0    1   531_cons    acattagtgctttattacgtggt     catttctttggcactttctgat   \n",
       "1    2   432_cons     acctgcattcgtatgtgttcta     ttcgaggtgggagattttcttc   \n",
       "2    3    473_var     tgctatggtacctaaacctacg       cgatgggtcaaacggaatcg   \n",
       "3    4    193_var     gccatgacgatatctacaatgc     gcgaacgctgattctaattttg   \n",
       "4    2    126_var       ttcaggagctcaccaaatcg     tccttcacctgttgtactgtat   \n",
       "5    3    416_var     aactggcactagtcatgtacat     caattaattccgttagcccgtg   \n",
       "6    4    495_var      ttcctcgcacactcatgtaag       tctcgatgtagaactgcacg   \n",
       "7    5    435_var     agttctttggagagttcggaat     aagaaaagaagtgagaaagcga   \n",
       "8    6    283_var    cacacaacttttatgtttaatca      aaaataatggccactattccc   \n",
       "9   10   205_cons      agacacaaggccttgaagaag     acagcttcttcttgtcatcgta   \n",
       "10   8      579_X         cacctgacactcgatggg         catgcagccggctacttg   \n",
       "11  10      575_X      agactttcaggccatgatacc     tcagtgaacgttgacttggtaa   \n",
       "12  17      576_X     aggagatgcaagttactatcgc       cactatcgtagcccaggttc   \n",
       "13  18      582_X         gaggcgcaggacacgatc      ctcctggatcttctgcatctc   \n",
       "14  22      587_X     tttcagcagaaacatccacatg     gtttcgatgtacttggtggttt   \n",
       "15   1  474_other    tgaattatgattaatggtgtgcc       caagtgggtgtgacattcca   \n",
       "16   3  433_other     aatgcttccattaactgccatt   ggttaagttttaatgtgcgtaaca   \n",
       "17   4  298_other  tagaattacaatcaagtggaagacg      ttatacagtggatggcagtcg   \n",
       "18   5  356_other     tctatcaccatgcacttcttgt     aatgtagataggctaaaaccgc   \n",
       "19   6  439_other     gcgcgtaatcgttttatcaaga    acaaatctgattacgatatgatc   \n",
       "20   7  269_other     aatgattcggatgatttgcttc     ccgtcattatttcccaatcaaa   \n",
       "21   9  140_other   gtgaattagattttaatgaagtaa  agtgggcagatttaattttagatct   \n",
       "22  10  245_other     cgcaattgtcacaaatctgtat       gcagtttacggcttttctcc   \n",
       "23  12   65_other       gcaaaatttccgtcccatta      tgtaattagctgtgtcttgtg   \n",
       "\n",
       "    FpTm(Celsius degree)  RpTm(Celsius degree)  FpGC(%)  RpGC(%)  FpPos  \\\n",
       "0                  56.87                 56.10    34.78    36.36     18   \n",
       "1                  58.12                 58.65    40.91    45.46      5   \n",
       "2                  57.66                 59.91    45.46    55.00     46   \n",
       "3                  57.98                 57.58    45.46    40.91     16   \n",
       "4                  58.48                 57.08    50.00    40.91    123   \n",
       "5                  57.77                 58.63    40.91    45.46     19   \n",
       "6                  58.62                 58.02    47.62    50.00     68   \n",
       "7                  57.97                 56.82    40.91    36.36     48   \n",
       "8                  52.53                 54.45    26.09    38.10     53   \n",
       "9                  58.73                 58.07    47.62    40.91     41   \n",
       "10                 57.65                 59.46    61.11    61.11      0   \n",
       "11                 58.03                 58.46    47.62    40.91     11   \n",
       "12                 58.60                 58.06    45.46    55.00     24   \n",
       "13                 61.14                 58.19    66.67    52.38     24   \n",
       "14                 58.08                 57.76    40.91    40.91      6   \n",
       "15                 55.92                 58.61    34.78    50.00     13   \n",
       "16                 57.50                 57.05    36.36    33.33     23   \n",
       "17                 57.76                 58.13    36.00    47.62     52   \n",
       "18                 58.04                 56.89    40.91    40.91     40   \n",
       "19                 58.39                 52.88    40.91    30.44     47   \n",
       "20                 56.06                 55.63    36.36    36.36     12   \n",
       "21                 50.09                 57.16    20.83    32.00      2   \n",
       "22                 56.29                 57.95    36.36    50.00      8   \n",
       "23                 55.11                 54.46    40.00    38.10      4   \n",
       "\n",
       "    RpPos  Penalty  AmpSize(bp) SNPs in primer?  AlnID  \\\n",
       "0     207     1.90          190            None    531   \n",
       "1     217     0.65          213            None    432   \n",
       "2     269     3.91          224            None    473   \n",
       "3     249     0.42          234            None    193   \n",
       "4     314     0.92          192        low freq    126   \n",
       "5     212     0.63          194        low freq    416   \n",
       "6     261     2.02          194        low freq    495   \n",
       "7     242     1.18          195        low freq    435   \n",
       "8     249     4.55          197        low freq    283   \n",
       "9     275     0.07          235        low freq    205   \n",
       "10    191     5.46          192       high freq    579   \n",
       "11    202     0.46          192       high freq    575   \n",
       "12    221     2.06          198       high freq    576   \n",
       "13    224     1.19          201       high freq    582   \n",
       "14    208     0.24          203       high freq    587   \n",
       "15    202     2.61          190            None    474   \n",
       "16    212     2.95          190            None    433   \n",
       "17    241     1.13          190            None    298   \n",
       "18    239     1.11          200            None    356   \n",
       "19    247     6.12          201            None    439   \n",
       "20    214     2.37          203            None    269   \n",
       "21    217     3.84          216            None    140   \n",
       "22    230     2.05          223            None    245   \n",
       "23    235     4.54          232            None     65   \n",
       "\n",
       "                            fp_deg  \\\n",
       "0          ACATTAGTGCTTTATTACGTGGT   \n",
       "1           ACCTGCATTCGTATGTGTTCTA   \n",
       "2           TGCTATGGTACCTAAACCTACG   \n",
       "3           GCCATGACGATATCTACAATGC   \n",
       "4             TTCAGGAGCTCACCAAATCG   \n",
       "5           AACTgGCACTAGTCATGTACAT   \n",
       "6            TTCCTCGCACACTCATGTAAG   \n",
       "7           AGTTCTTTGGAGAGTTCgGAAT   \n",
       "8         CAC-ACAACTTTTATGTTTAATCA   \n",
       "9            AGACACAAGGCCTTGAAGAAG   \n",
       "10  [C/T]ACCTGACA[C/T]TCGAT[G/C]GG   \n",
       "11   aGA[C/T]TTTCAGGCCATGAT[A/C]CC   \n",
       "12      AGGA[G/A]ATGCAAGTTACTATcGC   \n",
       "13        GAGGC[G/A/T]CAGGACACgATc   \n",
       "14          TTtCAGCAGAAACATCCACATG   \n",
       "15         TGAATTATGATTAATGGTGTGCC   \n",
       "16          AATGCTTCCATTAACTGCCATT   \n",
       "17       TAGAATTACAATCAAGTGGAAGACG   \n",
       "18          TCTATCACCATGCACTTCTTGT   \n",
       "19          GCGCGTAATCGTTTTATCAAGA   \n",
       "20          AATGATTCGGATGATTTGCTTC   \n",
       "21        GTGAATTAGATTTTAATGAAGTAA   \n",
       "22       ---CGCAATTGTCACAAATCTGTAT   \n",
       "23            GCAAAATTTCCGTCCCATTA   \n",
       "\n",
       "                                               rp_deg  \n",
       "0                              CATTTCTTTGGCACTTTCTGAT  \n",
       "1                              TTCGAGGTGGGAGATTTTCTTC  \n",
       "2                                CGATGGGTCAAACGGAATCG  \n",
       "3                              GCGAACGCTGATTCTAATTTTG  \n",
       "4                              TCCTTCACCTGTTGTACTGTAT  \n",
       "5                              CAATTAATTCCGTTAGCCCGTG  \n",
       "6                                TCTCGATGTAGAACTGCACg  \n",
       "7                              AAGAAAAGAAGTGAGAAAGCGA  \n",
       "8                               AAAaTAATGGCCACTATTCCC  \n",
       "9                              ACAGCTTCTTCTTGTCaTCGTA  \n",
       "10  [C/-][A/C/-][T/-/C][G/-/C][C/-/T]AGCCGGCTA[C/T...  \n",
       "11             TC[A/C/T/G]GTGAA[C/G]GTTGA[C/T]TTgGTAA  \n",
       "12                       C[A/G]CTATCGTAGCCCAG[G/A]TTC  \n",
       "13                              cTCCTGGATCTTCTGCATCTC  \n",
       "14                         GT[T/C]TCGATGTACTTGGTGGTTT  \n",
       "15                               CAAGTGGGTGTGACATTCCA  \n",
       "16                           GGTTAAGTTTTAATGTGCGTAACA  \n",
       "17                              TTATACAGTGGATGGCAGTCG  \n",
       "18                             AATGTAGATAGGCTAAAACCGC  \n",
       "19                            ACAAATCTGATTACGATATGATC  \n",
       "20                             CCGTCATTATTTCCCAATCAAA  \n",
       "21                          AGTGGGCAGATTTAATTTTAGATCT  \n",
       "22                               GCAGTTTACGGCTTTTCTCC  \n",
       "23                              TGTAATTAGCTGTGTCTTGTG  "
      ]
     },
     "execution_count": 67,
     "metadata": {},
     "output_type": "execute_result"
    }
   ],
   "source": [
    "primers['Multiplex_core']['AlnID'] = primers['Multiplex_core']['TemplateID'] \\\n",
    "                                        .str.split('_').apply(lambda x: int(x[0]))\n",
    "# vectorize primer search, excluded - alignment list is passed entirely\n",
    "primers['Multiplex_core']['fp_deg'] = np.vectorize(get_primer, excluded=[0]) \\\n",
    "                                     (alns, primers['Multiplex_core']['AlnID'],\n",
    "                                      primers['Multiplex_core']['ForwardPrimer(Fp)'])\n",
    "primers['Multiplex_core']['rp_deg'] = np.vectorize(get_primer, excluded=[0]) \\\n",
    "                                     (alns, primers['Multiplex_core']['AlnID'],\n",
    "                                      primers['Multiplex_core']['ReversePrimer(Rp)'], reverse=True)\n",
    "primers['Multiplex_core']"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 69,
   "metadata": {},
   "outputs": [
    {
     "data": {
      "text/html": [
       "<div>\n",
       "<style scoped>\n",
       "    .dataframe tbody tr th:only-of-type {\n",
       "        vertical-align: middle;\n",
       "    }\n",
       "\n",
       "    .dataframe tbody tr th {\n",
       "        vertical-align: top;\n",
       "    }\n",
       "\n",
       "    .dataframe thead th {\n",
       "        text-align: right;\n",
       "    }\n",
       "</style>\n",
       "<table border=\"1\" class=\"dataframe\">\n",
       "  <thead>\n",
       "    <tr style=\"text-align: right;\">\n",
       "      <th></th>\n",
       "      <th>ID</th>\n",
       "      <th>TemplateID</th>\n",
       "      <th>ForwardPrimer(Fp)</th>\n",
       "      <th>ReversePrimer(Rp)</th>\n",
       "      <th>FpTm(Celsius degree)</th>\n",
       "      <th>RpTm(Celsius degree)</th>\n",
       "      <th>FpGC(%)</th>\n",
       "      <th>RpGC(%)</th>\n",
       "      <th>FpPos</th>\n",
       "      <th>RpPos</th>\n",
       "      <th>Penalty</th>\n",
       "      <th>AmpSize(bp)</th>\n",
       "      <th>SNPs in primer?</th>\n",
       "      <th>AlnID</th>\n",
       "      <th>fp_deg</th>\n",
       "      <th>rp_deg</th>\n",
       "    </tr>\n",
       "  </thead>\n",
       "  <tbody>\n",
       "    <tr>\n",
       "      <th>0</th>\n",
       "      <td>2</td>\n",
       "      <td>77_var</td>\n",
       "      <td>gtctcggagcacatcgtg</td>\n",
       "      <td>tcgtacttcattattctttggactg</td>\n",
       "      <td>58.47</td>\n",
       "      <td>57.76</td>\n",
       "      <td>61.11</td>\n",
       "      <td>36.00</td>\n",
       "      <td>9</td>\n",
       "      <td>198</td>\n",
       "      <td>3.24</td>\n",
       "      <td>190</td>\n",
       "      <td>high freq</td>\n",
       "      <td>77</td>\n",
       "      <td>GTcTCgGAGCACAT[C/T]GTG</td>\n",
       "      <td>TCGTACTTCATTATTCTTTGGACTG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>1</th>\n",
       "      <td>3</td>\n",
       "      <td>83_var</td>\n",
       "      <td>agtgactccagacggtgtt</td>\n",
       "      <td>ccaaggatttgctactaccact</td>\n",
       "      <td>58.76</td>\n",
       "      <td>58.37</td>\n",
       "      <td>52.63</td>\n",
       "      <td>45.46</td>\n",
       "      <td>11</td>\n",
       "      <td>200</td>\n",
       "      <td>0.37</td>\n",
       "      <td>190</td>\n",
       "      <td>high freq</td>\n",
       "      <td>83</td>\n",
       "      <td>AGTG[A/G]CTCCAGACGGTgTT</td>\n",
       "      <td>CC[A/T/G]AGGATTTGCTACTACCAcT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>2</th>\n",
       "      <td>4</td>\n",
       "      <td>174_var</td>\n",
       "      <td>gtctcgaaaccgtacgtactaa</td>\n",
       "      <td>cactactactggcctcggata</td>\n",
       "      <td>58.25</td>\n",
       "      <td>58.73</td>\n",
       "      <td>45.46</td>\n",
       "      <td>52.38</td>\n",
       "      <td>39</td>\n",
       "      <td>228</td>\n",
       "      <td>1.73</td>\n",
       "      <td>190</td>\n",
       "      <td>high freq</td>\n",
       "      <td>174</td>\n",
       "      <td>GTCTCGAAACCGTA[C/A]GT[A/G]CTAA</td>\n",
       "      <td>CACTAcTACTGGCC[T/C]CGGATA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>3</th>\n",
       "      <td>5</td>\n",
       "      <td>206_var</td>\n",
       "      <td>agacatatcaaccacaagcgta</td>\n",
       "      <td>acgaagctctgatatgtatcgt</td>\n",
       "      <td>58.12</td>\n",
       "      <td>57.48</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>14</td>\n",
       "      <td>204</td>\n",
       "      <td>0.52</td>\n",
       "      <td>191</td>\n",
       "      <td>high freq</td>\n",
       "      <td>206</td>\n",
       "      <td>AGACATATC[-/A]AA[C/A]-C[A/-]CAAGCGTA</td>\n",
       "      <td>a[C/T][G/T][A/T/G][A/G]GCTCTGATATGt[A/G]TCGT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>4</th>\n",
       "      <td>6</td>\n",
       "      <td>31_var</td>\n",
       "      <td>tgtgtacggtctgaagaacatc</td>\n",
       "      <td>ttatccggctccaagttaagg</td>\n",
       "      <td>58.67</td>\n",
       "      <td>58.04</td>\n",
       "      <td>45.46</td>\n",
       "      <td>47.62</td>\n",
       "      <td>5</td>\n",
       "      <td>195</td>\n",
       "      <td>1.04</td>\n",
       "      <td>191</td>\n",
       "      <td>high freq</td>\n",
       "      <td>31</td>\n",
       "      <td>TGT[G/C]TACGGTCTGAAGAACATc</td>\n",
       "      <td>TT[A/G]TCCGGCTCCAAGTTAAGG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>5</th>\n",
       "      <td>7</td>\n",
       "      <td>281_cons</td>\n",
       "      <td>ccttgtgaacactgttgaactt</td>\n",
       "      <td>agctgtttgaatcgaaaccttc</td>\n",
       "      <td>57.88</td>\n",
       "      <td>58.09</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>19</td>\n",
       "      <td>209</td>\n",
       "      <td>0.09</td>\n",
       "      <td>191</td>\n",
       "      <td>high freq</td>\n",
       "      <td>281</td>\n",
       "      <td>CCTTGTGAACACTGTTGAACTT</td>\n",
       "      <td>AgCTGTT[T/C]GAATCGAAACC[T/A]TC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>6</th>\n",
       "      <td>9</td>\n",
       "      <td>220_var</td>\n",
       "      <td>ggtcgtaaccaaatgtccaaag</td>\n",
       "      <td>gagtgcactgaaaacctatgtg</td>\n",
       "      <td>58.42</td>\n",
       "      <td>58.17</td>\n",
       "      <td>45.46</td>\n",
       "      <td>45.46</td>\n",
       "      <td>14</td>\n",
       "      <td>205</td>\n",
       "      <td>0.17</td>\n",
       "      <td>192</td>\n",
       "      <td>high freq</td>\n",
       "      <td>220</td>\n",
       "      <td>gGT[C/T]GTAACCAA[A/G]TGTCCAAAG</td>\n",
       "      <td>GAGTGCACTGAAAACCTATGTG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>7</th>\n",
       "      <td>11</td>\n",
       "      <td>418_cons</td>\n",
       "      <td>tgttacggttaccaaatatgct</td>\n",
       "      <td>ggattcttttcagaagacgctc</td>\n",
       "      <td>56.40</td>\n",
       "      <td>57.98</td>\n",
       "      <td>36.36</td>\n",
       "      <td>45.46</td>\n",
       "      <td>2</td>\n",
       "      <td>193</td>\n",
       "      <td>0.02</td>\n",
       "      <td>192</td>\n",
       "      <td>high freq</td>\n",
       "      <td>418</td>\n",
       "      <td>TGTTACG[G/A]TT[A/G]CCAAAtATgCT</td>\n",
       "      <td>GGATTCTTTTCAGAaGACG[C/T]TC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>8</th>\n",
       "      <td>12</td>\n",
       "      <td>264_cons</td>\n",
       "      <td>cagatcctccgccttgatg</td>\n",
       "      <td>tccaagggtacgattgaaatca</td>\n",
       "      <td>58.27</td>\n",
       "      <td>58.03</td>\n",
       "      <td>57.90</td>\n",
       "      <td>40.91</td>\n",
       "      <td>23</td>\n",
       "      <td>214</td>\n",
       "      <td>0.03</td>\n",
       "      <td>192</td>\n",
       "      <td>high freq</td>\n",
       "      <td>264</td>\n",
       "      <td>CAGATC[C/T/G]TC[C/T/G]G[C/G]CTTGATg</td>\n",
       "      <td>TC[C/G]AAGGGTACgATTGAAATCA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>9</th>\n",
       "      <td>13</td>\n",
       "      <td>112_cons</td>\n",
       "      <td>gtatgtagcggggtccattg</td>\n",
       "      <td>gtgctatcgttaagaggtcgta</td>\n",
       "      <td>58.70</td>\n",
       "      <td>58.04</td>\n",
       "      <td>55.00</td>\n",
       "      <td>45.46</td>\n",
       "      <td>9</td>\n",
       "      <td>201</td>\n",
       "      <td>0.03</td>\n",
       "      <td>193</td>\n",
       "      <td>high freq</td>\n",
       "      <td>112</td>\n",
       "      <td>G[T/A]ATGTAGCG[G/A/T]GGTCCATTG</td>\n",
       "      <td>GTGCTATCGTTAAGAGGTCGTA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>10</th>\n",
       "      <td>14</td>\n",
       "      <td>382_cons</td>\n",
       "      <td>acaagagcaagtactgggagta</td>\n",
       "      <td>atcagctcggtgaacttctc</td>\n",
       "      <td>59.09</td>\n",
       "      <td>57.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>50.00</td>\n",
       "      <td>4</td>\n",
       "      <td>199</td>\n",
       "      <td>2.09</td>\n",
       "      <td>196</td>\n",
       "      <td>high freq</td>\n",
       "      <td>382</td>\n",
       "      <td>ACAAGAGCAAGTAcTGGGAGTa</td>\n",
       "      <td>ATCAGCTC[G/A/C]GTGAACTTCTC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>11</th>\n",
       "      <td>15</td>\n",
       "      <td>345_var</td>\n",
       "      <td>gtgttttgtcgaatcatttcaca</td>\n",
       "      <td>cagctgattgtactctgtgagt</td>\n",
       "      <td>57.13</td>\n",
       "      <td>58.40</td>\n",
       "      <td>34.78</td>\n",
       "      <td>45.46</td>\n",
       "      <td>2</td>\n",
       "      <td>198</td>\n",
       "      <td>0.40</td>\n",
       "      <td>197</td>\n",
       "      <td>high freq</td>\n",
       "      <td>345</td>\n",
       "      <td>GTg--TTTTGTCGAATCATTTCAcA</td>\n",
       "      <td>CAGCTGATTGT[A/C/G]CTCTGTGAGT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>12</th>\n",
       "      <td>16</td>\n",
       "      <td>317_var</td>\n",
       "      <td>catgttccacaaggaggaaag</td>\n",
       "      <td>atagaacgagtagatatcgcgg</td>\n",
       "      <td>57.38</td>\n",
       "      <td>57.72</td>\n",
       "      <td>47.62</td>\n",
       "      <td>45.46</td>\n",
       "      <td>17</td>\n",
       "      <td>214</td>\n",
       "      <td>0.28</td>\n",
       "      <td>198</td>\n",
       "      <td>high freq</td>\n",
       "      <td>317</td>\n",
       "      <td>cATGTTCCAcAAGGAGGAAAG</td>\n",
       "      <td>[A/G]TAGAACGAGTAgAT[A/G]TCGCG[G/A]</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>13</th>\n",
       "      <td>19</td>\n",
       "      <td>117_cons</td>\n",
       "      <td>ttagcttgctcgatctgatctt</td>\n",
       "      <td>atacttagcaggaccttcatcg</td>\n",
       "      <td>57.93</td>\n",
       "      <td>58.00</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>109</td>\n",
       "      <td>309</td>\n",
       "      <td>0.00</td>\n",
       "      <td>201</td>\n",
       "      <td>high freq</td>\n",
       "      <td>117</td>\n",
       "      <td>TTAG[C/T][T/C]TGCTCGATcTGATCTT</td>\n",
       "      <td>ATACTTAGCAGGACCTTCAT[C/A/T]G</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>14</th>\n",
       "      <td>20</td>\n",
       "      <td>238_cons</td>\n",
       "      <td>atgggtgtgaacatggagac</td>\n",
       "      <td>catgtcggtaaggatgaccag</td>\n",
       "      <td>58.09</td>\n",
       "      <td>58.75</td>\n",
       "      <td>50.00</td>\n",
       "      <td>52.38</td>\n",
       "      <td>30</td>\n",
       "      <td>230</td>\n",
       "      <td>1.75</td>\n",
       "      <td>201</td>\n",
       "      <td>high freq</td>\n",
       "      <td>238</td>\n",
       "      <td>ATGGGTGTGAACATGGAGAC</td>\n",
       "      <td>CATGTCGGT[A/C/G]AGgATGAC[C/G]AG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>15</th>\n",
       "      <td>21</td>\n",
       "      <td>500_var</td>\n",
       "      <td>cccgctaatgaaaagtaatggt</td>\n",
       "      <td>ccaacaaacggtgcaatatttg</td>\n",
       "      <td>57.34</td>\n",
       "      <td>57.93</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>1</td>\n",
       "      <td>203</td>\n",
       "      <td>0.07</td>\n",
       "      <td>203</td>\n",
       "      <td>high freq</td>\n",
       "      <td>500</td>\n",
       "      <td>[C/-][C/T]CGcTAATGAAAAGTAATGG[T/A]</td>\n",
       "      <td>CCAACAAACGG[T/G][G/A/C]CAATATTTG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>16</th>\n",
       "      <td>23</td>\n",
       "      <td>87_cons</td>\n",
       "      <td>tcaaccaggacaagaagaacat</td>\n",
       "      <td>cagatcagcgagatgaccttc</td>\n",
       "      <td>58.16</td>\n",
       "      <td>58.83</td>\n",
       "      <td>40.91</td>\n",
       "      <td>52.38</td>\n",
       "      <td>10</td>\n",
       "      <td>214</td>\n",
       "      <td>1.83</td>\n",
       "      <td>205</td>\n",
       "      <td>high freq</td>\n",
       "      <td>87</td>\n",
       "      <td>TCAACCAGGACAAGAAGAACAT</td>\n",
       "      <td>CA[G/A]ATCAGCGAGAT[G/A/C]ACCTT[C/G]</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>17</th>\n",
       "      <td>24</td>\n",
       "      <td>177_cons</td>\n",
       "      <td>gccttaggggatattcaactgt</td>\n",
       "      <td>aatacactttaacgtaaggcgc</td>\n",
       "      <td>58.16</td>\n",
       "      <td>57.99</td>\n",
       "      <td>45.46</td>\n",
       "      <td>40.91</td>\n",
       "      <td>87</td>\n",
       "      <td>291</td>\n",
       "      <td>0.02</td>\n",
       "      <td>205</td>\n",
       "      <td>high freq</td>\n",
       "      <td>177</td>\n",
       "      <td>GCCTTAGG[G/T]GATAT[T/A]CAACTGT</td>\n",
       "      <td>[A/G]ATACACTTTAACGTAAGGCGC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>18</th>\n",
       "      <td>25</td>\n",
       "      <td>214_cons</td>\n",
       "      <td>gttgagaacaacgaagacaacc</td>\n",
       "      <td>atcagatcgacgacaccctt</td>\n",
       "      <td>58.64</td>\n",
       "      <td>59.11</td>\n",
       "      <td>45.46</td>\n",
       "      <td>50.00</td>\n",
       "      <td>0</td>\n",
       "      <td>205</td>\n",
       "      <td>3.11</td>\n",
       "      <td>206</td>\n",
       "      <td>high freq</td>\n",
       "      <td>214</td>\n",
       "      <td>GTtGAGAACAACGA[A/G]GACAACC</td>\n",
       "      <td>ATCAGATCGAC[G/A]ACACCcTT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>19</th>\n",
       "      <td>26</td>\n",
       "      <td>293_cons</td>\n",
       "      <td>aagtgatcggacggtatgtg</td>\n",
       "      <td>actcattcctttaagctagcgt</td>\n",
       "      <td>58.00</td>\n",
       "      <td>58.12</td>\n",
       "      <td>50.00</td>\n",
       "      <td>40.91</td>\n",
       "      <td>24</td>\n",
       "      <td>232</td>\n",
       "      <td>0.12</td>\n",
       "      <td>209</td>\n",
       "      <td>high freq</td>\n",
       "      <td>293</td>\n",
       "      <td>AAGTG[A/G]TCgGACGGTATGTG</td>\n",
       "      <td>ACTCATTCCTTTAA[G/A]CTAGCgT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>20</th>\n",
       "      <td>27</td>\n",
       "      <td>449_cons</td>\n",
       "      <td>tgtatgaatccttccttgtggt</td>\n",
       "      <td>tttgggaagttcctatggtgat</td>\n",
       "      <td>57.67</td>\n",
       "      <td>57.66</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>18</td>\n",
       "      <td>228</td>\n",
       "      <td>0.34</td>\n",
       "      <td>211</td>\n",
       "      <td>high freq</td>\n",
       "      <td>449</td>\n",
       "      <td>TGTATGAAtCC[T/C]TCCTTGTGGT</td>\n",
       "      <td>TTTGGGA[A/G]GT[T/G]CCTATGGTGAT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>21</th>\n",
       "      <td>30</td>\n",
       "      <td>99_var</td>\n",
       "      <td>gcgttgttcgttgtagttctac</td>\n",
       "      <td>cgggatatggaagatcacaact</td>\n",
       "      <td>58.53</td>\n",
       "      <td>58.25</td>\n",
       "      <td>45.46</td>\n",
       "      <td>45.46</td>\n",
       "      <td>37</td>\n",
       "      <td>250</td>\n",
       "      <td>0.25</td>\n",
       "      <td>214</td>\n",
       "      <td>high freq</td>\n",
       "      <td>99</td>\n",
       "      <td>GCGT[T/C]GTTCGTTGTAGTTCTAC</td>\n",
       "      <td>CG[G/C]GA[T/C]ATGGAAGA[T/C]CACAA[C/T]T</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>22</th>\n",
       "      <td>31</td>\n",
       "      <td>128_var</td>\n",
       "      <td>atttcattgcctgtgccatatc</td>\n",
       "      <td>caaaatgtctaccgtcgacaag</td>\n",
       "      <td>57.99</td>\n",
       "      <td>58.26</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>19</td>\n",
       "      <td>232</td>\n",
       "      <td>0.26</td>\n",
       "      <td>214</td>\n",
       "      <td>high freq</td>\n",
       "      <td>128</td>\n",
       "      <td>ATTTCATtGCCTGTGCCATATC</td>\n",
       "      <td>CAA[A/C]ATGTCTACCGTCGACAAG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>23</th>\n",
       "      <td>32</td>\n",
       "      <td>325_var</td>\n",
       "      <td>ccgggaaatgaatttatcgaaga</td>\n",
       "      <td>cgagttgacccattttgtgaat</td>\n",
       "      <td>57.58</td>\n",
       "      <td>58.08</td>\n",
       "      <td>39.13</td>\n",
       "      <td>40.91</td>\n",
       "      <td>0</td>\n",
       "      <td>214</td>\n",
       "      <td>0.08</td>\n",
       "      <td>215</td>\n",
       "      <td>high freq</td>\n",
       "      <td>325</td>\n",
       "      <td>CC[G/C/T]GG[A/C]AATGA[A/G]TT[T/C]ATCGAAGA</td>\n",
       "      <td>CGA[G/A]TTGACCCA[T/C]TTTGTGAAT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>24</th>\n",
       "      <td>34</td>\n",
       "      <td>1_cons</td>\n",
       "      <td>aaggtggaatcacagcatatga</td>\n",
       "      <td>gcgggaagataaggaacgattt</td>\n",
       "      <td>58.09</td>\n",
       "      <td>59.38</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>55</td>\n",
       "      <td>271</td>\n",
       "      <td>1.38</td>\n",
       "      <td>217</td>\n",
       "      <td>high freq</td>\n",
       "      <td>1</td>\n",
       "      <td>AAGGTGGAATCACAGCATATGA</td>\n",
       "      <td>[G/A]CGGGAAGA[T/C]AA[G/A]GA[A/G]CGATTT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>25</th>\n",
       "      <td>35</td>\n",
       "      <td>516_var</td>\n",
       "      <td>tttggcgtctgatagaacatct</td>\n",
       "      <td>agctccacggacagtctatata</td>\n",
       "      <td>58.12</td>\n",
       "      <td>58.23</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.46</td>\n",
       "      <td>18</td>\n",
       "      <td>234</td>\n",
       "      <td>0.23</td>\n",
       "      <td>217</td>\n",
       "      <td>high freq</td>\n",
       "      <td>516</td>\n",
       "      <td>TTTGGCGTCTG[A/G]TAGAACATCT</td>\n",
       "      <td>AGCT[C/T]CACGGACAGTCTAT[A/C]TA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>26</th>\n",
       "      <td>36</td>\n",
       "      <td>508_cons</td>\n",
       "      <td>cttcatgaacagacggttgtg</td>\n",
       "      <td>atggtgcagtgtatcatcgag</td>\n",
       "      <td>58.04</td>\n",
       "      <td>58.48</td>\n",
       "      <td>47.62</td>\n",
       "      <td>47.62</td>\n",
       "      <td>21</td>\n",
       "      <td>239</td>\n",
       "      <td>1.48</td>\n",
       "      <td>219</td>\n",
       "      <td>high freq</td>\n",
       "      <td>508</td>\n",
       "      <td>CTTCATgAACAGACGGTTGTG</td>\n",
       "      <td>ATGGTGCAGTGTATCAT[C/T]GAg</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>27</th>\n",
       "      <td>38</td>\n",
       "      <td>200_cons</td>\n",
       "      <td>gttccctcgaagcatactttg</td>\n",
       "      <td>gctacgttgctaccactagtaa</td>\n",
       "      <td>57.55</td>\n",
       "      <td>58.22</td>\n",
       "      <td>47.62</td>\n",
       "      <td>45.46</td>\n",
       "      <td>81</td>\n",
       "      <td>303</td>\n",
       "      <td>0.22</td>\n",
       "      <td>223</td>\n",
       "      <td>high freq</td>\n",
       "      <td>200</td>\n",
       "      <td>GTTC[C/A/T]CTCGAAGC[A/C]TACTTTG</td>\n",
       "      <td>GCTAC[G/A]TTgCTACC[A/G]CTAGTAA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>28</th>\n",
       "      <td>39</td>\n",
       "      <td>277_cons</td>\n",
       "      <td>cagcacgttggagagtcc</td>\n",
       "      <td>gtgcaacttccgtgtaatagtc</td>\n",
       "      <td>58.29</td>\n",
       "      <td>57.99</td>\n",
       "      <td>61.11</td>\n",
       "      <td>45.46</td>\n",
       "      <td>2</td>\n",
       "      <td>227</td>\n",
       "      <td>0.01</td>\n",
       "      <td>226</td>\n",
       "      <td>high freq</td>\n",
       "      <td>277</td>\n",
       "      <td>CAGCACGTT[G/C]GAgAGTCC</td>\n",
       "      <td>GTGCAACTTC[C/T]GTGTAATAGTC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>29</th>\n",
       "      <td>40</td>\n",
       "      <td>72_cons</td>\n",
       "      <td>gaacttctccttcgcgtcc</td>\n",
       "      <td>tatactggttcacggttgagtt</td>\n",
       "      <td>58.82</td>\n",
       "      <td>57.72</td>\n",
       "      <td>57.90</td>\n",
       "      <td>40.91</td>\n",
       "      <td>92</td>\n",
       "      <td>318</td>\n",
       "      <td>0.29</td>\n",
       "      <td>227</td>\n",
       "      <td>high freq</td>\n",
       "      <td>72</td>\n",
       "      <td>GAACTTCTCCTTCGC[G/A]TCC</td>\n",
       "      <td>T[A/C/G]TACTGGTTCACGGT[T/C]GAGTT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>...</th>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "      <td>...</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>39</th>\n",
       "      <td>55</td>\n",
       "      <td>337_cons</td>\n",
       "      <td>gacctgcatcctgtacgtg</td>\n",
       "      <td>cacttgaccttactccagcc</td>\n",
       "      <td>58.52</td>\n",
       "      <td>58.48</td>\n",
       "      <td>57.90</td>\n",
       "      <td>55.00</td>\n",
       "      <td>32</td>\n",
       "      <td>280</td>\n",
       "      <td>2.48</td>\n",
       "      <td>249</td>\n",
       "      <td>high freq</td>\n",
       "      <td>337</td>\n",
       "      <td>GAC[C/G]TGCATcCTGTACGTG</td>\n",
       "      <td>CACTTGACCTTACTCCAGCC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>40</th>\n",
       "      <td>1</td>\n",
       "      <td>399_other</td>\n",
       "      <td>aaaagtttcaaaattaatcattcag</td>\n",
       "      <td>agtgcaagacagatggctgaaa</td>\n",
       "      <td>52.42</td>\n",
       "      <td>61.08</td>\n",
       "      <td>20.00</td>\n",
       "      <td>45.46</td>\n",
       "      <td>5</td>\n",
       "      <td>194</td>\n",
       "      <td>3.08</td>\n",
       "      <td>190</td>\n",
       "      <td>low freq</td>\n",
       "      <td>399</td>\n",
       "      <td>AAAAGTTTCAAAATTAATCATTCAG</td>\n",
       "      <td>AG-TGCAAGACAGATgGCtGAAA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>41</th>\n",
       "      <td>2</td>\n",
       "      <td>33_other</td>\n",
       "      <td>gagcgtgcggccaagatg</td>\n",
       "      <td>acagaccgacgttaatggc</td>\n",
       "      <td>62.43</td>\n",
       "      <td>58.44</td>\n",
       "      <td>66.67</td>\n",
       "      <td>52.63</td>\n",
       "      <td>39</td>\n",
       "      <td>228</td>\n",
       "      <td>3.44</td>\n",
       "      <td>190</td>\n",
       "      <td>low freq</td>\n",
       "      <td>33</td>\n",
       "      <td>GAGCGtGCGGCcAAGATG</td>\n",
       "      <td>ACAgACCGACGTTAATGGC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>42</th>\n",
       "      <td>3</td>\n",
       "      <td>442_other</td>\n",
       "      <td>tcaatttgtcatctttgtttacca</td>\n",
       "      <td>attgacatcaatcgatcgttca</td>\n",
       "      <td>56.06</td>\n",
       "      <td>56.84</td>\n",
       "      <td>29.17</td>\n",
       "      <td>36.36</td>\n",
       "      <td>16</td>\n",
       "      <td>205</td>\n",
       "      <td>1.16</td>\n",
       "      <td>190</td>\n",
       "      <td>low freq</td>\n",
       "      <td>442</td>\n",
       "      <td>TCAATTTGTCaTCTTTGTTTACCA</td>\n",
       "      <td>ATTGACATCAATCGATCGTTCA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>43</th>\n",
       "      <td>4</td>\n",
       "      <td>253_other</td>\n",
       "      <td>taaaatgattgcacacctgctcgc</td>\n",
       "      <td>aatcgcgtcggacttgtg</td>\n",
       "      <td>63.26</td>\n",
       "      <td>58.70</td>\n",
       "      <td>45.83</td>\n",
       "      <td>55.56</td>\n",
       "      <td>26</td>\n",
       "      <td>215</td>\n",
       "      <td>4.70</td>\n",
       "      <td>190</td>\n",
       "      <td>low freq</td>\n",
       "      <td>253</td>\n",
       "      <td>TAAAATGATTGCACACCTGcTCGC</td>\n",
       "      <td>AATCGcGTcGGACTTGTG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>44</th>\n",
       "      <td>6</td>\n",
       "      <td>567_other</td>\n",
       "      <td>aatacgctcgcctatagaatgt</td>\n",
       "      <td>aatgcaaattatgtgcgtcgta</td>\n",
       "      <td>57.80</td>\n",
       "      <td>57.97</td>\n",
       "      <td>40.91</td>\n",
       "      <td>36.36</td>\n",
       "      <td>49</td>\n",
       "      <td>238</td>\n",
       "      <td>0.03</td>\n",
       "      <td>190</td>\n",
       "      <td>low freq</td>\n",
       "      <td>567</td>\n",
       "      <td>AATACGCTCGCCTATAGAATGT</td>\n",
       "      <td>AATGCAAATTATGTgcgTCGTA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>45</th>\n",
       "      <td>9</td>\n",
       "      <td>81_other</td>\n",
       "      <td>cgctgccagataccgtgc</td>\n",
       "      <td>ctaccaacaccgtctggag</td>\n",
       "      <td>61.82</td>\n",
       "      <td>57.76</td>\n",
       "      <td>66.67</td>\n",
       "      <td>57.90</td>\n",
       "      <td>21</td>\n",
       "      <td>210</td>\n",
       "      <td>3.24</td>\n",
       "      <td>190</td>\n",
       "      <td>low freq</td>\n",
       "      <td>81</td>\n",
       "      <td>CGCTGCCAGATaCCGTGC</td>\n",
       "      <td>CTACCAAcACCGTCTGGAG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>46</th>\n",
       "      <td>10</td>\n",
       "      <td>47_other</td>\n",
       "      <td>gatataaattgtcgatcacacaaact</td>\n",
       "      <td>tgcatttatcgtagtacaatctca</td>\n",
       "      <td>57.02</td>\n",
       "      <td>56.63</td>\n",
       "      <td>30.77</td>\n",
       "      <td>33.33</td>\n",
       "      <td>17</td>\n",
       "      <td>207</td>\n",
       "      <td>3.37</td>\n",
       "      <td>191</td>\n",
       "      <td>low freq</td>\n",
       "      <td>47</td>\n",
       "      <td>GaTATAAATTGTCGATCACACAAACT</td>\n",
       "      <td>TGCATTTATCGTAGTACAATCTCA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>47</th>\n",
       "      <td>11</td>\n",
       "      <td>568_other</td>\n",
       "      <td>gattttattacaattttccttatga</td>\n",
       "      <td>accagaaattaaaatatgttacacttga</td>\n",
       "      <td>51.01</td>\n",
       "      <td>57.05</td>\n",
       "      <td>20.00</td>\n",
       "      <td>25.00</td>\n",
       "      <td>7</td>\n",
       "      <td>197</td>\n",
       "      <td>6.95</td>\n",
       "      <td>191</td>\n",
       "      <td>low freq</td>\n",
       "      <td>568</td>\n",
       "      <td>GATTTTATTACAaTTTTCCTTATGA</td>\n",
       "      <td>ACCAGAAATTAAAATATGTTAcACTTGA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>48</th>\n",
       "      <td>14</td>\n",
       "      <td>59_other</td>\n",
       "      <td>gctggcgcataattatcacaaa</td>\n",
       "      <td>tttccacttcatcgctcgc</td>\n",
       "      <td>58.62</td>\n",
       "      <td>59.11</td>\n",
       "      <td>40.91</td>\n",
       "      <td>52.63</td>\n",
       "      <td>20</td>\n",
       "      <td>211</td>\n",
       "      <td>4.11</td>\n",
       "      <td>192</td>\n",
       "      <td>low freq</td>\n",
       "      <td>59</td>\n",
       "      <td>GCTGGCGCATAATTATCaCAAA</td>\n",
       "      <td>tTTCCACTTCATCGCTCGC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>49</th>\n",
       "      <td>15</td>\n",
       "      <td>159_other</td>\n",
       "      <td>gacattttaattgatttactgtgtctc</td>\n",
       "      <td>ccatccatttcaatcaacgtca</td>\n",
       "      <td>56.45</td>\n",
       "      <td>57.88</td>\n",
       "      <td>29.63</td>\n",
       "      <td>40.91</td>\n",
       "      <td>21</td>\n",
       "      <td>213</td>\n",
       "      <td>0.12</td>\n",
       "      <td>193</td>\n",
       "      <td>low freq</td>\n",
       "      <td>159</td>\n",
       "      <td>GACATTTTAATTGATtTACTGTGTCTC</td>\n",
       "      <td>CCATCCATTTCAATCAACGTCA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>50</th>\n",
       "      <td>16</td>\n",
       "      <td>583_other</td>\n",
       "      <td>tgataatgaacggtgtgaatcg</td>\n",
       "      <td>gtgtcatttaactcaattgtaacttca</td>\n",
       "      <td>57.46</td>\n",
       "      <td>57.65</td>\n",
       "      <td>40.91</td>\n",
       "      <td>29.63</td>\n",
       "      <td>19</td>\n",
       "      <td>211</td>\n",
       "      <td>5.35</td>\n",
       "      <td>193</td>\n",
       "      <td>low freq</td>\n",
       "      <td>583</td>\n",
       "      <td>TGATAATGAaCGGTGTGAATCG</td>\n",
       "      <td>GTGTCATTTAACTCAATTGTAACTTCA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>51</th>\n",
       "      <td>19</td>\n",
       "      <td>355_other</td>\n",
       "      <td>acattgttggtaattaatgtcca</td>\n",
       "      <td>gctgtttatttgtgtatgcaaaca</td>\n",
       "      <td>55.10</td>\n",
       "      <td>57.82</td>\n",
       "      <td>30.44</td>\n",
       "      <td>33.33</td>\n",
       "      <td>11</td>\n",
       "      <td>205</td>\n",
       "      <td>2.18</td>\n",
       "      <td>195</td>\n",
       "      <td>low freq</td>\n",
       "      <td>355</td>\n",
       "      <td>ACATTGTTGGTAATTAATGTcCA</td>\n",
       "      <td>GCTGTtTATTTGTGTATGCAAACA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>52</th>\n",
       "      <td>21</td>\n",
       "      <td>44_other</td>\n",
       "      <td>cagtcaaatttccagacaatct</td>\n",
       "      <td>cggaagtgcatttgaaggaaaa</td>\n",
       "      <td>55.23</td>\n",
       "      <td>58.61</td>\n",
       "      <td>36.36</td>\n",
       "      <td>40.91</td>\n",
       "      <td>4</td>\n",
       "      <td>198</td>\n",
       "      <td>0.61</td>\n",
       "      <td>195</td>\n",
       "      <td>low freq</td>\n",
       "      <td>44</td>\n",
       "      <td>CAGTCAAATTTCCAGACAATCT</td>\n",
       "      <td>CGGAAGTGCATTTGAAGG-AAaA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>53</th>\n",
       "      <td>22</td>\n",
       "      <td>469_other</td>\n",
       "      <td>tgaaagcgtgcccaatttg</td>\n",
       "      <td>agtagctcattaatacaatcgct</td>\n",
       "      <td>57.99</td>\n",
       "      <td>56.23</td>\n",
       "      <td>47.37</td>\n",
       "      <td>34.78</td>\n",
       "      <td>10</td>\n",
       "      <td>205</td>\n",
       "      <td>2.77</td>\n",
       "      <td>196</td>\n",
       "      <td>low freq</td>\n",
       "      <td>469</td>\n",
       "      <td>TGAAAGCGTGCCCAATTTG</td>\n",
       "      <td>AgTAGCTCATTAAtACAATCGCt</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>54</th>\n",
       "      <td>23</td>\n",
       "      <td>182_other</td>\n",
       "      <td>gcgtactacgacatccgg</td>\n",
       "      <td>tagatgccctgcttgtagtt</td>\n",
       "      <td>57.95</td>\n",
       "      <td>56.82</td>\n",
       "      <td>61.11</td>\n",
       "      <td>45.00</td>\n",
       "      <td>26</td>\n",
       "      <td>222</td>\n",
       "      <td>3.18</td>\n",
       "      <td>197</td>\n",
       "      <td>low freq</td>\n",
       "      <td>182</td>\n",
       "      <td>GCGTACTACGACATCCGg</td>\n",
       "      <td>TAGATGCCCTGCTTGTAGTT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>55</th>\n",
       "      <td>25</td>\n",
       "      <td>167_other</td>\n",
       "      <td>tccagctcctcgatgctc</td>\n",
       "      <td>aagcagcagaacaacgagat</td>\n",
       "      <td>59.04</td>\n",
       "      <td>58.40</td>\n",
       "      <td>61.11</td>\n",
       "      <td>45.00</td>\n",
       "      <td>12</td>\n",
       "      <td>208</td>\n",
       "      <td>2.40</td>\n",
       "      <td>197</td>\n",
       "      <td>low freq</td>\n",
       "      <td>167</td>\n",
       "      <td>TCCAGCTCcTCGATGCTC</td>\n",
       "      <td>AAGCAGCAGAACAAcGAgAT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>56</th>\n",
       "      <td>27</td>\n",
       "      <td>395_other</td>\n",
       "      <td>aaagttgatgaaaacgcgatga</td>\n",
       "      <td>tttgcgctctatttctgtcgat</td>\n",
       "      <td>58.10</td>\n",
       "      <td>59.06</td>\n",
       "      <td>36.36</td>\n",
       "      <td>40.91</td>\n",
       "      <td>5</td>\n",
       "      <td>204</td>\n",
       "      <td>1.06</td>\n",
       "      <td>200</td>\n",
       "      <td>low freq</td>\n",
       "      <td>395</td>\n",
       "      <td>AAAGTTGATGAAAACGCGATGA</td>\n",
       "      <td>TTTGCGCTCTATTTCTGTCGAT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>57</th>\n",
       "      <td>28</td>\n",
       "      <td>485_other</td>\n",
       "      <td>ttgaagatttattagaaatggg</td>\n",
       "      <td>cgaaagtatcaatcactgtcat</td>\n",
       "      <td>50.45</td>\n",
       "      <td>54.90</td>\n",
       "      <td>27.27</td>\n",
       "      <td>36.36</td>\n",
       "      <td>14</td>\n",
       "      <td>213</td>\n",
       "      <td>3.10</td>\n",
       "      <td>200</td>\n",
       "      <td>low freq</td>\n",
       "      <td>485</td>\n",
       "      <td>TtGAAGATTTATTAGAAATGGG</td>\n",
       "      <td>cGAAaGTATCAATCACTGTCAT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>58</th>\n",
       "      <td>29</td>\n",
       "      <td>280_other</td>\n",
       "      <td>cagaaatgactagaagcagtgc</td>\n",
       "      <td>acattacaatggaagtgatggc</td>\n",
       "      <td>57.98</td>\n",
       "      <td>57.60</td>\n",
       "      <td>45.46</td>\n",
       "      <td>40.91</td>\n",
       "      <td>53</td>\n",
       "      <td>253</td>\n",
       "      <td>0.40</td>\n",
       "      <td>201</td>\n",
       "      <td>low freq</td>\n",
       "      <td>280</td>\n",
       "      <td>CAGAAATGACTAGAAGCAGTGC</td>\n",
       "      <td>ACATTACAATGGAAGTGAtGGC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>59</th>\n",
       "      <td>30</td>\n",
       "      <td>413_other</td>\n",
       "      <td>caaatggcggtcgttgtt</td>\n",
       "      <td>ccgagggtgacacactttt</td>\n",
       "      <td>57.21</td>\n",
       "      <td>58.18</td>\n",
       "      <td>50.00</td>\n",
       "      <td>52.63</td>\n",
       "      <td>12</td>\n",
       "      <td>212</td>\n",
       "      <td>3.18</td>\n",
       "      <td>201</td>\n",
       "      <td>low freq</td>\n",
       "      <td>413</td>\n",
       "      <td>CAAATGGCGGtCGTTGTT</td>\n",
       "      <td>CCGAGGGTGACACACTTTT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>60</th>\n",
       "      <td>31</td>\n",
       "      <td>306_other</td>\n",
       "      <td>ataaaagcttcctcgacagct</td>\n",
       "      <td>tcatgtttataacaaattagtcaagct</td>\n",
       "      <td>57.97</td>\n",
       "      <td>56.74</td>\n",
       "      <td>42.86</td>\n",
       "      <td>25.93</td>\n",
       "      <td>6</td>\n",
       "      <td>207</td>\n",
       "      <td>6.26</td>\n",
       "      <td>202</td>\n",
       "      <td>low freq</td>\n",
       "      <td>306</td>\n",
       "      <td>ATAAAAGCTTCcTCGACAGCT</td>\n",
       "      <td>TCATGTTTATAACAAATTAGTCAAGCT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>61</th>\n",
       "      <td>32</td>\n",
       "      <td>526_other</td>\n",
       "      <td>taatttattaattggcgcaaagca</td>\n",
       "      <td>atgatgaatggaaccgcaca</td>\n",
       "      <td>57.13</td>\n",
       "      <td>58.46</td>\n",
       "      <td>29.17</td>\n",
       "      <td>45.00</td>\n",
       "      <td>18</td>\n",
       "      <td>222</td>\n",
       "      <td>2.46</td>\n",
       "      <td>205</td>\n",
       "      <td>low freq</td>\n",
       "      <td>526</td>\n",
       "      <td>tAATTTATTAATTGGcGCAaAGCA</td>\n",
       "      <td>ATGATGAATGGAACCGCACA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>62</th>\n",
       "      <td>33</td>\n",
       "      <td>268_other</td>\n",
       "      <td>gcgaccacccacaaattaatta</td>\n",
       "      <td>gtgaagcaaatcatccgaatca</td>\n",
       "      <td>57.88</td>\n",
       "      <td>57.96</td>\n",
       "      <td>40.91</td>\n",
       "      <td>40.91</td>\n",
       "      <td>1</td>\n",
       "      <td>211</td>\n",
       "      <td>0.04</td>\n",
       "      <td>211</td>\n",
       "      <td>low freq</td>\n",
       "      <td>268</td>\n",
       "      <td>GCGACCACCCACAAATTAATTA</td>\n",
       "      <td>GtGAAGCAAATCATCCGAATCA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>63</th>\n",
       "      <td>36</td>\n",
       "      <td>297_other</td>\n",
       "      <td>accatgatgtgctaatctgctt</td>\n",
       "      <td>gcggtgattttattgatccc</td>\n",
       "      <td>58.70</td>\n",
       "      <td>55.38</td>\n",
       "      <td>40.91</td>\n",
       "      <td>45.00</td>\n",
       "      <td>13</td>\n",
       "      <td>227</td>\n",
       "      <td>4.63</td>\n",
       "      <td>215</td>\n",
       "      <td>low freq</td>\n",
       "      <td>297</td>\n",
       "      <td>aCCATGATGTGCTAATCTGCTT</td>\n",
       "      <td>GcGGTGATTTTATTGATCCC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>64</th>\n",
       "      <td>39</td>\n",
       "      <td>555_other</td>\n",
       "      <td>cagctcagcaaggccaac</td>\n",
       "      <td>caacctcgagctgcagatc</td>\n",
       "      <td>59.61</td>\n",
       "      <td>58.89</td>\n",
       "      <td>61.11</td>\n",
       "      <td>57.90</td>\n",
       "      <td>10</td>\n",
       "      <td>229</td>\n",
       "      <td>3.89</td>\n",
       "      <td>220</td>\n",
       "      <td>low freq</td>\n",
       "      <td>555</td>\n",
       "      <td>CAGCTCAGCAAGGCCAAC</td>\n",
       "      <td>CaACcTCgAGCTGCAGATC</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>65</th>\n",
       "      <td>40</td>\n",
       "      <td>586_other</td>\n",
       "      <td>aagacagctccaatcaaca</td>\n",
       "      <td>caaagtcatggcccattaataa</td>\n",
       "      <td>54.85</td>\n",
       "      <td>55.33</td>\n",
       "      <td>42.11</td>\n",
       "      <td>36.36</td>\n",
       "      <td>2</td>\n",
       "      <td>222</td>\n",
       "      <td>2.67</td>\n",
       "      <td>221</td>\n",
       "      <td>low freq</td>\n",
       "      <td>586</td>\n",
       "      <td>AaGACAGCTCCAATCAACA</td>\n",
       "      <td>cAAAGTCATGGCCCATTAATAA</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>66</th>\n",
       "      <td>43</td>\n",
       "      <td>252_other</td>\n",
       "      <td>gctcgtactcggccagtat</td>\n",
       "      <td>tcgatcgtatcctgaaactgtt</td>\n",
       "      <td>59.54</td>\n",
       "      <td>57.87</td>\n",
       "      <td>57.90</td>\n",
       "      <td>40.91</td>\n",
       "      <td>34</td>\n",
       "      <td>258</td>\n",
       "      <td>0.13</td>\n",
       "      <td>225</td>\n",
       "      <td>low freq</td>\n",
       "      <td>252</td>\n",
       "      <td>GCTCGTACTCGGccAGTAT</td>\n",
       "      <td>TCGATCGTATCCTGAAACTGTT</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>67</th>\n",
       "      <td>45</td>\n",
       "      <td>249_other</td>\n",
       "      <td>gtaggcccatcgcaccgg</td>\n",
       "      <td>gatcgagagctacatcgagaag</td>\n",
       "      <td>63.48</td>\n",
       "      <td>58.51</td>\n",
       "      <td>72.22</td>\n",
       "      <td>50.00</td>\n",
       "      <td>46</td>\n",
       "      <td>278</td>\n",
       "      <td>0.51</td>\n",
       "      <td>233</td>\n",
       "      <td>low freq</td>\n",
       "      <td>249</td>\n",
       "      <td>GTAGGCCCATCGCACCGG</td>\n",
       "      <td>GATCGAgAGCTACATCGAGAAG</td>\n",
       "    </tr>\n",
       "    <tr>\n",
       "      <th>68</th>\n",
       "      <td>48</td>\n",
       "      <td>164_other</td>\n",
       "      <td>gatgccaaaattctgaacga</td>\n",
       "      <td>tcaaccacttcgacaagaacc</td>\n",
       "      <td>55.16</td>\n",
       "      <td>59.02</td>\n",
       "      <td>40.00</td>\n",
       "      <td>47.62</td>\n",
       "      <td>10</td>\n",
       "      <td>259</td>\n",
       "      <td>2.02</td>\n",
       "      <td>250</td>\n",
       "      <td>low freq</td>\n",
       "      <td>164</td>\n",
       "      <td>GATGCCAAAATTCTGAAcGA</td>\n",
       "      <td>TCAACCACTTCGACAAGAACC</td>\n",
       "    </tr>\n",
       "  </tbody>\n",
       "</table>\n",
       "<p>69 rows × 16 columns</p>\n",
       "</div>"
      ],
      "text/plain": [
       "    ID TemplateID            ForwardPrimer(Fp)             ReversePrimer(Rp)  \\\n",
       "0    2     77_var           gtctcggagcacatcgtg     tcgtacttcattattctttggactg   \n",
       "1    3     83_var          agtgactccagacggtgtt        ccaaggatttgctactaccact   \n",
       "2    4    174_var       gtctcgaaaccgtacgtactaa         cactactactggcctcggata   \n",
       "3    5    206_var       agacatatcaaccacaagcgta        acgaagctctgatatgtatcgt   \n",
       "4    6     31_var       tgtgtacggtctgaagaacatc         ttatccggctccaagttaagg   \n",
       "5    7   281_cons       ccttgtgaacactgttgaactt        agctgtttgaatcgaaaccttc   \n",
       "6    9    220_var       ggtcgtaaccaaatgtccaaag        gagtgcactgaaaacctatgtg   \n",
       "7   11   418_cons       tgttacggttaccaaatatgct        ggattcttttcagaagacgctc   \n",
       "8   12   264_cons          cagatcctccgccttgatg        tccaagggtacgattgaaatca   \n",
       "9   13   112_cons         gtatgtagcggggtccattg        gtgctatcgttaagaggtcgta   \n",
       "10  14   382_cons       acaagagcaagtactgggagta          atcagctcggtgaacttctc   \n",
       "11  15    345_var      gtgttttgtcgaatcatttcaca        cagctgattgtactctgtgagt   \n",
       "12  16    317_var        catgttccacaaggaggaaag        atagaacgagtagatatcgcgg   \n",
       "13  19   117_cons       ttagcttgctcgatctgatctt        atacttagcaggaccttcatcg   \n",
       "14  20   238_cons         atgggtgtgaacatggagac         catgtcggtaaggatgaccag   \n",
       "15  21    500_var       cccgctaatgaaaagtaatggt        ccaacaaacggtgcaatatttg   \n",
       "16  23    87_cons       tcaaccaggacaagaagaacat         cagatcagcgagatgaccttc   \n",
       "17  24   177_cons       gccttaggggatattcaactgt        aatacactttaacgtaaggcgc   \n",
       "18  25   214_cons       gttgagaacaacgaagacaacc          atcagatcgacgacaccctt   \n",
       "19  26   293_cons         aagtgatcggacggtatgtg        actcattcctttaagctagcgt   \n",
       "20  27   449_cons       tgtatgaatccttccttgtggt        tttgggaagttcctatggtgat   \n",
       "21  30     99_var       gcgttgttcgttgtagttctac        cgggatatggaagatcacaact   \n",
       "22  31    128_var       atttcattgcctgtgccatatc        caaaatgtctaccgtcgacaag   \n",
       "23  32    325_var      ccgggaaatgaatttatcgaaga        cgagttgacccattttgtgaat   \n",
       "24  34     1_cons       aaggtggaatcacagcatatga        gcgggaagataaggaacgattt   \n",
       "25  35    516_var       tttggcgtctgatagaacatct        agctccacggacagtctatata   \n",
       "26  36   508_cons        cttcatgaacagacggttgtg         atggtgcagtgtatcatcgag   \n",
       "27  38   200_cons        gttccctcgaagcatactttg        gctacgttgctaccactagtaa   \n",
       "28  39   277_cons           cagcacgttggagagtcc        gtgcaacttccgtgtaatagtc   \n",
       "29  40    72_cons          gaacttctccttcgcgtcc        tatactggttcacggttgagtt   \n",
       "..  ..        ...                          ...                           ...   \n",
       "39  55   337_cons          gacctgcatcctgtacgtg          cacttgaccttactccagcc   \n",
       "40   1  399_other    aaaagtttcaaaattaatcattcag        agtgcaagacagatggctgaaa   \n",
       "41   2   33_other           gagcgtgcggccaagatg           acagaccgacgttaatggc   \n",
       "42   3  442_other     tcaatttgtcatctttgtttacca        attgacatcaatcgatcgttca   \n",
       "43   4  253_other     taaaatgattgcacacctgctcgc            aatcgcgtcggacttgtg   \n",
       "44   6  567_other       aatacgctcgcctatagaatgt        aatgcaaattatgtgcgtcgta   \n",
       "45   9   81_other           cgctgccagataccgtgc           ctaccaacaccgtctggag   \n",
       "46  10   47_other   gatataaattgtcgatcacacaaact      tgcatttatcgtagtacaatctca   \n",
       "47  11  568_other    gattttattacaattttccttatga  accagaaattaaaatatgttacacttga   \n",
       "48  14   59_other       gctggcgcataattatcacaaa           tttccacttcatcgctcgc   \n",
       "49  15  159_other  gacattttaattgatttactgtgtctc        ccatccatttcaatcaacgtca   \n",
       "50  16  583_other       tgataatgaacggtgtgaatcg   gtgtcatttaactcaattgtaacttca   \n",
       "51  19  355_other      acattgttggtaattaatgtcca      gctgtttatttgtgtatgcaaaca   \n",
       "52  21   44_other       cagtcaaatttccagacaatct        cggaagtgcatttgaaggaaaa   \n",
       "53  22  469_other          tgaaagcgtgcccaatttg       agtagctcattaatacaatcgct   \n",
       "54  23  182_other           gcgtactacgacatccgg          tagatgccctgcttgtagtt   \n",
       "55  25  167_other           tccagctcctcgatgctc          aagcagcagaacaacgagat   \n",
       "56  27  395_other       aaagttgatgaaaacgcgatga        tttgcgctctatttctgtcgat   \n",
       "57  28  485_other       ttgaagatttattagaaatggg        cgaaagtatcaatcactgtcat   \n",
       "58  29  280_other       cagaaatgactagaagcagtgc        acattacaatggaagtgatggc   \n",
       "59  30  413_other           caaatggcggtcgttgtt           ccgagggtgacacactttt   \n",
       "60  31  306_other        ataaaagcttcctcgacagct   tcatgtttataacaaattagtcaagct   \n",
       "61  32  526_other     taatttattaattggcgcaaagca          atgatgaatggaaccgcaca   \n",
       "62  33  268_other       gcgaccacccacaaattaatta        gtgaagcaaatcatccgaatca   \n",
       "63  36  297_other       accatgatgtgctaatctgctt          gcggtgattttattgatccc   \n",
       "64  39  555_other           cagctcagcaaggccaac           caacctcgagctgcagatc   \n",
       "65  40  586_other          aagacagctccaatcaaca        caaagtcatggcccattaataa   \n",
       "66  43  252_other          gctcgtactcggccagtat        tcgatcgtatcctgaaactgtt   \n",
       "67  45  249_other           gtaggcccatcgcaccgg        gatcgagagctacatcgagaag   \n",
       "68  48  164_other         gatgccaaaattctgaacga         tcaaccacttcgacaagaacc   \n",
       "\n",
       "    FpTm(Celsius degree)  RpTm(Celsius degree)  FpGC(%)  RpGC(%)  FpPos  \\\n",
       "0                  58.47                 57.76    61.11    36.00      9   \n",
       "1                  58.76                 58.37    52.63    45.46     11   \n",
       "2                  58.25                 58.73    45.46    52.38     39   \n",
       "3                  58.12                 57.48    40.91    40.91     14   \n",
       "4                  58.67                 58.04    45.46    47.62      5   \n",
       "5                  57.88                 58.09    40.91    40.91     19   \n",
       "6                  58.42                 58.17    45.46    45.46     14   \n",
       "7                  56.40                 57.98    36.36    45.46      2   \n",
       "8                  58.27                 58.03    57.90    40.91     23   \n",
       "9                  58.70                 58.04    55.00    45.46      9   \n",
       "10                 59.09                 57.91    45.46    50.00      4   \n",
       "11                 57.13                 58.40    34.78    45.46      2   \n",
       "12                 57.38                 57.72    47.62    45.46     17   \n",
       "13                 57.93                 58.00    40.91    45.46    109   \n",
       "14                 58.09                 58.75    50.00    52.38     30   \n",
       "15                 57.34                 57.93    40.91    40.91      1   \n",
       "16                 58.16                 58.83    40.91    52.38     10   \n",
       "17                 58.16                 57.99    45.46    40.91     87   \n",
       "18                 58.64                 59.11    45.46    50.00      0   \n",
       "19                 58.00                 58.12    50.00    40.91     24   \n",
       "20                 57.67                 57.66    40.91    40.91     18   \n",
       "21                 58.53                 58.25    45.46    45.46     37   \n",
       "22                 57.99                 58.26    40.91    45.46     19   \n",
       "23                 57.58                 58.08    39.13    40.91      0   \n",
       "24                 58.09                 59.38    40.91    45.46     55   \n",
       "25                 58.12                 58.23    40.91    45.46     18   \n",
       "26                 58.04                 58.48    47.62    47.62     21   \n",
       "27                 57.55                 58.22    47.62    45.46     81   \n",
       "28                 58.29                 57.99    61.11    45.46      2   \n",
       "29                 58.82                 57.72    57.90    40.91     92   \n",
       "..                   ...                   ...      ...      ...    ...   \n",
       "39                 58.52                 58.48    57.90    55.00     32   \n",
       "40                 52.42                 61.08    20.00    45.46      5   \n",
       "41                 62.43                 58.44    66.67    52.63     39   \n",
       "42                 56.06                 56.84    29.17    36.36     16   \n",
       "43                 63.26                 58.70    45.83    55.56     26   \n",
       "44                 57.80                 57.97    40.91    36.36     49   \n",
       "45                 61.82                 57.76    66.67    57.90     21   \n",
       "46                 57.02                 56.63    30.77    33.33     17   \n",
       "47                 51.01                 57.05    20.00    25.00      7   \n",
       "48                 58.62                 59.11    40.91    52.63     20   \n",
       "49                 56.45                 57.88    29.63    40.91     21   \n",
       "50                 57.46                 57.65    40.91    29.63     19   \n",
       "51                 55.10                 57.82    30.44    33.33     11   \n",
       "52                 55.23                 58.61    36.36    40.91      4   \n",
       "53                 57.99                 56.23    47.37    34.78     10   \n",
       "54                 57.95                 56.82    61.11    45.00     26   \n",
       "55                 59.04                 58.40    61.11    45.00     12   \n",
       "56                 58.10                 59.06    36.36    40.91      5   \n",
       "57                 50.45                 54.90    27.27    36.36     14   \n",
       "58                 57.98                 57.60    45.46    40.91     53   \n",
       "59                 57.21                 58.18    50.00    52.63     12   \n",
       "60                 57.97                 56.74    42.86    25.93      6   \n",
       "61                 57.13                 58.46    29.17    45.00     18   \n",
       "62                 57.88                 57.96    40.91    40.91      1   \n",
       "63                 58.70                 55.38    40.91    45.00     13   \n",
       "64                 59.61                 58.89    61.11    57.90     10   \n",
       "65                 54.85                 55.33    42.11    36.36      2   \n",
       "66                 59.54                 57.87    57.90    40.91     34   \n",
       "67                 63.48                 58.51    72.22    50.00     46   \n",
       "68                 55.16                 59.02    40.00    47.62     10   \n",
       "\n",
       "    RpPos  Penalty  AmpSize(bp) SNPs in primer?  AlnID  \\\n",
       "0     198     3.24          190       high freq     77   \n",
       "1     200     0.37          190       high freq     83   \n",
       "2     228     1.73          190       high freq    174   \n",
       "3     204     0.52          191       high freq    206   \n",
       "4     195     1.04          191       high freq     31   \n",
       "5     209     0.09          191       high freq    281   \n",
       "6     205     0.17          192       high freq    220   \n",
       "7     193     0.02          192       high freq    418   \n",
       "8     214     0.03          192       high freq    264   \n",
       "9     201     0.03          193       high freq    112   \n",
       "10    199     2.09          196       high freq    382   \n",
       "11    198     0.40          197       high freq    345   \n",
       "12    214     0.28          198       high freq    317   \n",
       "13    309     0.00          201       high freq    117   \n",
       "14    230     1.75          201       high freq    238   \n",
       "15    203     0.07          203       high freq    500   \n",
       "16    214     1.83          205       high freq     87   \n",
       "17    291     0.02          205       high freq    177   \n",
       "18    205     3.11          206       high freq    214   \n",
       "19    232     0.12          209       high freq    293   \n",
       "20    228     0.34          211       high freq    449   \n",
       "21    250     0.25          214       high freq     99   \n",
       "22    232     0.26          214       high freq    128   \n",
       "23    214     0.08          215       high freq    325   \n",
       "24    271     1.38          217       high freq      1   \n",
       "25    234     0.23          217       high freq    516   \n",
       "26    239     1.48          219       high freq    508   \n",
       "27    303     0.22          223       high freq    200   \n",
       "28    227     0.01          226       high freq    277   \n",
       "29    318     0.29          227       high freq     72   \n",
       "..    ...      ...          ...             ...    ...   \n",
       "39    280     2.48          249       high freq    337   \n",
       "40    194     3.08          190        low freq    399   \n",
       "41    228     3.44          190        low freq     33   \n",
       "42    205     1.16          190        low freq    442   \n",
       "43    215     4.70          190        low freq    253   \n",
       "44    238     0.03          190        low freq    567   \n",
       "45    210     3.24          190        low freq     81   \n",
       "46    207     3.37          191        low freq     47   \n",
       "47    197     6.95          191        low freq    568   \n",
       "48    211     4.11          192        low freq     59   \n",
       "49    213     0.12          193        low freq    159   \n",
       "50    211     5.35          193        low freq    583   \n",
       "51    205     2.18          195        low freq    355   \n",
       "52    198     0.61          195        low freq     44   \n",
       "53    205     2.77          196        low freq    469   \n",
       "54    222     3.18          197        low freq    182   \n",
       "55    208     2.40          197        low freq    167   \n",
       "56    204     1.06          200        low freq    395   \n",
       "57    213     3.10          200        low freq    485   \n",
       "58    253     0.40          201        low freq    280   \n",
       "59    212     3.18          201        low freq    413   \n",
       "60    207     6.26          202        low freq    306   \n",
       "61    222     2.46          205        low freq    526   \n",
       "62    211     0.04          211        low freq    268   \n",
       "63    227     4.63          215        low freq    297   \n",
       "64    229     3.89          220        low freq    555   \n",
       "65    222     2.67          221        low freq    586   \n",
       "66    258     0.13          225        low freq    252   \n",
       "67    278     0.51          233        low freq    249   \n",
       "68    259     2.02          250        low freq    164   \n",
       "\n",
       "                                       fp_deg  \\\n",
       "0                      GTcTCgGAGCACAT[C/T]GTG   \n",
       "1                     AGTG[A/G]CTCCAGACGGTgTT   \n",
       "2              GTCTCGAAACCGTA[C/A]GT[A/G]CTAA   \n",
       "3        AGACATATC[-/A]AA[C/A]-C[A/-]CAAGCGTA   \n",
       "4                  TGT[G/C]TACGGTCTGAAGAACATc   \n",
       "5                      CCTTGTGAACACTGTTGAACTT   \n",
       "6              gGT[C/T]GTAACCAA[A/G]TGTCCAAAG   \n",
       "7              TGTTACG[G/A]TT[A/G]CCAAAtATgCT   \n",
       "8         CAGATC[C/T/G]TC[C/T/G]G[C/G]CTTGATg   \n",
       "9              G[T/A]ATGTAGCG[G/A/T]GGTCCATTG   \n",
       "10                     ACAAGAGCAAGTAcTGGGAGTa   \n",
       "11                  GTg--TTTTGTCGAATCATTTCAcA   \n",
       "12                      cATGTTCCAcAAGGAGGAAAG   \n",
       "13             TTAG[C/T][T/C]TGCTCGATcTGATCTT   \n",
       "14                       ATGGGTGTGAACATGGAGAC   \n",
       "15         [C/-][C/T]CGcTAATGAAAAGTAATGG[T/A]   \n",
       "16                     TCAACCAGGACAAGAAGAACAT   \n",
       "17             GCCTTAGG[G/T]GATAT[T/A]CAACTGT   \n",
       "18                 GTtGAGAACAACGA[A/G]GACAACC   \n",
       "19                   AAGTG[A/G]TCgGACGGTATGTG   \n",
       "20                 TGTATGAAtCC[T/C]TCCTTGTGGT   \n",
       "21                 GCGT[T/C]GTTCGTTGTAGTTCTAC   \n",
       "22                     ATTTCATtGCCTGTGCCATATC   \n",
       "23  CC[G/C/T]GG[A/C]AATGA[A/G]TT[T/C]ATCGAAGA   \n",
       "24                     AAGGTGGAATCACAGCATATGA   \n",
       "25                 TTTGGCGTCTG[A/G]TAGAACATCT   \n",
       "26                      CTTCATgAACAGACGGTTGTG   \n",
       "27            GTTC[C/A/T]CTCGAAGC[A/C]TACTTTG   \n",
       "28                     CAGCACGTT[G/C]GAgAGTCC   \n",
       "29                    GAACTTCTCCTTCGC[G/A]TCC   \n",
       "..                                        ...   \n",
       "39                    GAC[C/G]TGCATcCTGTACGTG   \n",
       "40                  AAAAGTTTCAAAATTAATCATTCAG   \n",
       "41                         GAGCGtGCGGCcAAGATG   \n",
       "42                   TCAATTTGTCaTCTTTGTTTACCA   \n",
       "43                   TAAAATGATTGCACACCTGcTCGC   \n",
       "44                     AATACGCTCGCCTATAGAATGT   \n",
       "45                         CGCTGCCAGATaCCGTGC   \n",
       "46                 GaTATAAATTGTCGATCACACAAACT   \n",
       "47                  GATTTTATTACAaTTTTCCTTATGA   \n",
       "48                     GCTGGCGCATAATTATCaCAAA   \n",
       "49                GACATTTTAATTGATtTACTGTGTCTC   \n",
       "50                     TGATAATGAaCGGTGTGAATCG   \n",
       "51                    ACATTGTTGGTAATTAATGTcCA   \n",
       "52                     CAGTCAAATTTCCAGACAATCT   \n",
       "53                        TGAAAGCGTGCCCAATTTG   \n",
       "54                         GCGTACTACGACATCCGg   \n",
       "55                         TCCAGCTCcTCGATGCTC   \n",
       "56                     AAAGTTGATGAAAACGCGATGA   \n",
       "57                     TtGAAGATTTATTAGAAATGGG   \n",
       "58                     CAGAAATGACTAGAAGCAGTGC   \n",
       "59                         CAAATGGCGGtCGTTGTT   \n",
       "60                      ATAAAAGCTTCcTCGACAGCT   \n",
       "61                   tAATTTATTAATTGGcGCAaAGCA   \n",
       "62                     GCGACCACCCACAAATTAATTA   \n",
       "63                     aCCATGATGTGCTAATCTGCTT   \n",
       "64                         CAGCTCAGCAAGGCCAAC   \n",
       "65                        AaGACAGCTCCAATCAACA   \n",
       "66                        GCTCGTACTCGGccAGTAT   \n",
       "67                         GTAGGCCCATCGCACCGG   \n",
       "68                       GATGCCAAAATTCTGAAcGA   \n",
       "\n",
       "                                          rp_deg  \n",
       "0                      TCGTACTTCATTATTCTTTGGACTG  \n",
       "1                   CC[A/T/G]AGGATTTGCTACTACCAcT  \n",
       "2                      CACTAcTACTGGCC[T/C]CGGATA  \n",
       "3   a[C/T][G/T][A/T/G][A/G]GCTCTGATATGt[A/G]TCGT  \n",
       "4                      TT[A/G]TCCGGCTCCAAGTTAAGG  \n",
       "5                 AgCTGTT[T/C]GAATCGAAACC[T/A]TC  \n",
       "6                         GAGTGCACTGAAAACCTATGTG  \n",
       "7                     GGATTCTTTTCAGAaGACG[C/T]TC  \n",
       "8                     TC[C/G]AAGGGTACgATTGAAATCA  \n",
       "9                         GTGCTATCGTTAAGAGGTCGTA  \n",
       "10                    ATCAGCTC[G/A/C]GTGAACTTCTC  \n",
       "11                  CAGCTGATTGT[A/C/G]CTCTGTGAGT  \n",
       "12            [A/G]TAGAACGAGTAgAT[A/G]TCGCG[G/A]  \n",
       "13                  ATACTTAGCAGGACCTTCAT[C/A/T]G  \n",
       "14               CATGTCGGT[A/C/G]AGgATGAC[C/G]AG  \n",
       "15              CCAACAAACGG[T/G][G/A/C]CAATATTTG  \n",
       "16           CA[G/A]ATCAGCGAGAT[G/A/C]ACCTT[C/G]  \n",
       "17                    [A/G]ATACACTTTAACGTAAGGCGC  \n",
       "18                      ATCAGATCGAC[G/A]ACACCcTT  \n",
       "19                    ACTCATTCCTTTAA[G/A]CTAGCgT  \n",
       "20                TTTGGGA[A/G]GT[T/G]CCTATGGTGAT  \n",
       "21        CG[G/C]GA[T/C]ATGGAAGA[T/C]CACAA[C/T]T  \n",
       "22                    CAA[A/C]ATGTCTACCGTCGACAAG  \n",
       "23                CGA[G/A]TTGACCCA[T/C]TTTGTGAAT  \n",
       "24        [G/A]CGGGAAGA[T/C]AA[G/A]GA[A/G]CGATTT  \n",
       "25                AGCT[C/T]CACGGACAGTCTAT[A/C]TA  \n",
       "26                     ATGGTGCAGTGTATCAT[C/T]GAg  \n",
       "27                GCTAC[G/A]TTgCTACC[A/G]CTAGTAA  \n",
       "28                    GTGCAACTTC[C/T]GTGTAATAGTC  \n",
       "29              T[A/C/G]TACTGGTTCACGGT[T/C]GAGTT  \n",
       "..                                           ...  \n",
       "39                          CACTTGACCTTACTCCAGCC  \n",
       "40                       AG-TGCAAGACAGATgGCtGAAA  \n",
       "41                           ACAgACCGACGTTAATGGC  \n",
       "42                        ATTGACATCAATCGATCGTTCA  \n",
       "43                            AATCGcGTcGGACTTGTG  \n",
       "44                        AATGCAAATTATGTgcgTCGTA  \n",
       "45                           CTACCAAcACCGTCTGGAG  \n",
       "46                      TGCATTTATCGTAGTACAATCTCA  \n",
       "47                  ACCAGAAATTAAAATATGTTAcACTTGA  \n",
       "48                           tTTCCACTTCATCGCTCGC  \n",
       "49                        CCATCCATTTCAATCAACGTCA  \n",
       "50                   GTGTCATTTAACTCAATTGTAACTTCA  \n",
       "51                      GCTGTtTATTTGTGTATGCAAACA  \n",
       "52                       CGGAAGTGCATTTGAAGG-AAaA  \n",
       "53                       AgTAGCTCATTAAtACAATCGCt  \n",
       "54                          TAGATGCCCTGCTTGTAGTT  \n",
       "55                          AAGCAGCAGAACAAcGAgAT  \n",
       "56                        TTTGCGCTCTATTTCTGTCGAT  \n",
       "57                        cGAAaGTATCAATCACTGTCAT  \n",
       "58                        ACATTACAATGGAAGTGAtGGC  \n",
       "59                           CCGAGGGTGACACACTTTT  \n",
       "60                   TCATGTTTATAACAAATTAGTCAAGCT  \n",
       "61                          ATGATGAATGGAACCGCACA  \n",
       "62                        GtGAAGCAAATCATCCGAATCA  \n",
       "63                          GcGGTGATTTTATTGATCCC  \n",
       "64                           CaACcTCgAGCTGCAGATC  \n",
       "65                        cAAAGTCATGGCCCATTAATAA  \n",
       "66                        TCGATCGTATCCTGAAACTGTT  \n",
       "67                        GATCGAgAGCTACATCGAGAAG  \n",
       "68                         TCAACCACTTCGACAAGAACC  \n",
       "\n",
       "[69 rows x 16 columns]"
      ]
     },
     "execution_count": 69,
     "metadata": {},
     "output_type": "execute_result"
    }
   ],
   "source": [
    "primers['Additional markers to add']\n",
    "primers['Additional markers to add']['AlnID'] = primers['Additional markers to add']['TemplateID'] \\\n",
    "                                        .str.split('_').apply(lambda x: int(x[0]))\n",
    "# vectorize primer search, excluded - alignment list is passed entirely\n",
    "primers['Additional markers to add']['fp_deg'] = np.vectorize(get_primer, excluded=[0]) \\\n",
    "                                     (alns, primers['Additional markers to add']['AlnID'],\n",
    "                                      primers['Additional markers to add']['ForwardPrimer(Fp)'])\n",
    "primers['Additional markers to add']['rp_deg'] = np.vectorize(get_primer, excluded=[0]) \\\n",
    "                                     (alns, primers['Additional markers to add']['AlnID'],\n",
    "                                      primers['Additional markers to add']['ReversePrimer(Rp)'], reverse=True)\n",
    "primers['Additional markers to add']"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": 75,
   "metadata": {},
   "outputs": [],
   "source": [
    "writer = pd.ExcelWriter(OUT_FILE)\n",
    "for (k,v) in primers.items():\n",
    "    v.to_excel(writer, sheet_name=k)\n",
    "writer.save()"
   ]
  }
 ],
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