-
Notifications
You must be signed in to change notification settings - Fork 8
/
Copy pathsalmon-tximport-deseq2-v0.3.smk
276 lines (246 loc) · 9.95 KB
/
salmon-tximport-deseq2-v0.3.smk
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
from os.path import join, isfile
from itertools import combinations
include: "rules/common.smk"
# configfile: 'config.yml'
workdir: config['workdir']
################### globals #############################################
# Full path to an uncompressed FASTA file with all chromosome sequences.
CDNA = config['cdna']
# Full path to a folder that holds all of your FASTQ files.
FASTQ_DIR = config['fastq_dir']
READ_LEN = config['read_length']
PAIRED = config['paired']
# Full path to a Genome.
GENOME = config['genome']
#CDNA = join(GENOME,"gencode.v25.transcripts.fa")
# genome sequence
FASTA_REF = config['dna']
# index_dir
SALMON_INDEX_DIR=config['salmon_index']
# index basename
INDEX_PREFIX = 'hg38'
# gtf
GTF_FILE = config['gtf']
GTF_Genes = GTF_FILE.rstrip(".gtf")+".extracted.genes.annotation.txt"
GTF_Trans = GTF_FILE.rstrip(".gtf")+".extracted.transx2gene.txt"
############ Samples ##################
# A Snakemake regular expression matching the forward mate FASTQ files.
# the part in curly brackets {} will be saved, so the variable SAMPLES
# is a list of strings #['Sample1','Sample2'].
#notice that SAMPLES, has a trailing comma.
#you must include this trailing comma, or else the code won’t work correctly.
#SAMPLES, = glob_wildcards(join(FASTQ_DIR, '{sample, SRR[^/]+}_R1.fastq.gz'))
SAMPLES,SAMPLES_ALIAS,GROUP,TIME = parse_samples(config['sample_meta'])
uGroup=unique(GROUP)
# Patterns for the 1st mate and the 2nd mate using the 'sample' wildcard.
PATTERN_R1 = config['read_pattern']['r1']
PATTERN_R2 = config['read_pattern']['r2']
# dirs
DIRS = ['qc','mapped','alternative_splicing', 'gene_expression',
'differential_expression','logs','temp']
# go domain
GO_DOMAIN = config['enrichr_library']
# cutoff
LOG2FC = config['log2fc']
FDR = config['fdr']
########### Target output files #################
SALMON_INDEX = expand(SALMON_INDEX_DIR+"/{prefix}.bin", prefix=['pos','mphf'])
SALMON_QUANT_Trans = expand("salmon/{sample}/quant.sf", sample=SAMPLES)
SALMON_QUANT_Genes = expand("salmon/{sample}/quant.genes.sf", sample=SAMPLES)
# RAW_COUNTS ="quant/sample.raw.counts.txt"
SAMPLE_TPM_ANNO = "quant/gene_expression.TPM.annotated.csv"
SAMPLE_TXTPM_ANNO ="quant/transcripts_expression.TPM.annotated.csv"
SALMON_OBJ= "quant/txi.salmon.RData"
DESEQ_DDS = "quant/deseq2.dds.RData"
DESEQ_NTD = "quant/deseq2.ntd.Rdata"
DESEQ_RES = ["differential_expression/diff_{t}_vs_{c}/diff_{t}_vs_{c}_results.txt".format(t=j, c=i)
for i, j in combinations(uGroup, 2)]
DESEQ_ANNO = [res.replace(".txt", ".annotated.xls") for res in DESEQ_RES]
DESEQ_HEATMAP = ["differential_expression/diff_{t}_vs_{c}/diff_{t}_vs_{c}_all.degs.pdf".format(t=j, c=i)
for i, j in combinations(uGroup, 2)]
GSEA_RES=["differential_expression/GO/GSEA_{treat}_vs_{ctrl}/%s/gseapy.gsea.gene_set.report.csv"%domain for domain in GO_DOMAIN]
GSEA_FINAL=["differential_expression/GO/GSEA_%s_vs_%s/KEGG_2016/gseapy.gsea.gene_set.report.csv"%(j, i) for i, j in combinations(uGroup, 2)]
#Enrichr = ["GO/Enrichr_{treat}_vs_{ctrl}/{domain}_{types}/{domain}.{type}.enrichr.reports.txt",type=["all","up","down"]
#GSEA_OUT = [ GSEA_RES.format(treat=uGroup[i], ctrl=uGroup[i-1]) for i in range(1, len(uGroup))]
################## Rules #######################################
rule target:
input: DESEQ_DDS, #DESEQ_ANNO,
SAMPLE_TPM_ANNO, SAMPLE_TXTPM_ANNO,
#DESEQ_RES, DESEQ_HEATMAP, GSEA_FINAL
rule salmon_index:
input: CDNA
output:
bin = SALMON_INDEX,
#fa = join(SALMON_INDEX_DIR,"ref_k31_fixed.fa")
threads: 8
params:
genome_dir=config['genome'],
cdna=CDNA,
outdir=SALMON_INDEX_DIR,
extra=" --gencode "
shell:
"salmon index {params.extra} -t {input} -i {params.outdir}"
#"salmon index -i /genome/{params.outdir} -t /genome/{params.cdna} {params.extra}"
########## notes on salmon quant ###################################################################
### <LIBTYPE>
### A (automatically infer the library type)
### IU (an unstranded paired-end library where the reads face each other)
### SF (a stranded single-end protocol where the reads come from the forward strand)
### more types visit: http://salmon.readthedocs.io/en/latest/salmon.html#quasi-mapping-based-mode-including-lightweight-alignment
"""
salmon quant
-l (depends on the lib type, ISR for truseq stranded, equivalent to tophat -fr-firststrand)
-p (the number of available cores on the instance)
-g (the gene level counts will be part of output)
--incompatPrior 0 (we don’t want reads incompatible with the library type)
--fldMean 250 (for single-ended reads only, kallisto default, can change if there is info about lib prep)
--fldSD 25 (for single-ended reads only, kallisto default, can change if there is info about lib prep)
--numBootstraps 100 (maybe good for samples without technical replicates)
--seqBias (this option not for single-end )
--gcBias (this option not for single-end )
--writeUnmappedNames
Note:
Choose not to use --useVBOpt based on documentation and this link
https://groups.google.com/forum/#!topic/sailfish-users/-LBZD4aoJSc
The behavior will be more like Kallisto and RSEM instead of BitSeq
"""
############ salmon quant start ####################################################################
rule salmon_quant:
input:
index=SALMON_INDEX,
#index_dir=SALMON_INDEX_DIR,
gtf=GTF_FILE,
r1=join(FASTQ_DIR, PATTERN_R1),
r2=join(FASTQ_DIR, PATTERN_R2)
output:
sf="salmon/{sample}/quant.sf",
#outdir=directory("salmon/{sample}") # Directories as outputs
threads: 8
params:
r1=join(FASTQ_DIR, PATTERN_R1),
r2=join(FASTQ_DIR, PATTERN_R2),
index_dir=SALMON_INDEX_DIR,
outdir=join(config['workdir'],"salmon/{sample}"),
extra_paried=" --incompatPrior 0 --numBootstraps 100 --seqBias --gcBias --writeUnmappedNames",
#extra_single=" --fldMean 250 --fldSD 25 --incompatPrior 0 --numBootstraps 100 --writeUnmappedNames"
log: "logs/salmon/{sample}_salmons_quant.log"
shell:
"salmon quant -l A -i {params.index_dir} -1 {params.r1} -2 {params.r2} "
"-p {threads} -g {input.gtf} -o {params.outdir} {params.extra_paried} &> {log}"
rule tximport:
'''used for kallisto, Salmon, Sailfish, and RSEM. see:
http://bioconductor.org/packages/release/bioc/vignettes/tximport/inst/doc/tximport.html
###
good tutorial to look:
https://github.com/crazyhottommy/RNA-seq-analysis/blob/master/salmon_kalliso_STAR_compare.md#counts-versus-tpmrpkmfpkm
'''
input:
quant=expand("salmon/{sample}/quant.sf", sample=SAMPLES),
tx2gene=GTF_Trans
output:
tpm=temp("quant/gene_expression.TPM.txt"),
txtpm=temp("quant/transcripts_expression.TPM.txt"),
#counts=RAW_COUNTS,
image=SALMON_OBJ
params:
ids =",".join(SAMPLES)
threads: 1
script:
"scripts/runTximport.R"
# rule salmon_quantmerge:
# input:
# expand("salmon/{sample}/quant.sf", sample=SAMPLES),
# expand("salmon/{sample}/quant.genes.sf", sample=SAMPLES)
# output:
# tpm=SAMPLE_TPM,
# txtpm=SAMPLE_TXTPM,
# counts=RAW_COUNTS
# params:
# ids =" ".join(SAMPLES)
# shell:
# """salmon quantmerge --quants {params.ids} --genes -o {output.tpm}
# salmon quantmerge --quants {params.ids} --genes -c numreads -o {output.counts}
# salmon quantmerge --quants {params.ids} -o {output.txtpm}
# """
rule deseq2:
input:
image="quant/txi.salmon.RData",#SALMON_TPM
output:
res=DESEQ_RES,
ddsimage="quant/deseq2.dds.RData", #DESEQ_DDS
ntdimage="quant/deseq2.ntd.RData", #DESEQ_NTD
params:
group=" ".join(GROUP),#used for grouping each sample, to dectect degs.
time=" ".join(TIME),
alias=" ".join(SAMPLES_ALIAS)
threads: 8
script:
"scripts/runDESeq2.R"
rule gtf_extract:
input: GTF_FILE
output:
gene_anno=GTF_Genes,
tx2gene = GTF_Trans
script:
"scripts/extractGTF.py"
rule anno_DEGs:
input:
"differential_expression/diff_{treat}_vs_{ctrl}/diff_{treat}_vs_{ctrl}_results.txt"
output:
"differential_expression/diff_{treat}_vs_{ctrl}/diff_{treat}_vs_{ctrl}_results.annotated.xls"
params:
gene_anno=GTF_Genes,
tpm=SAMPLE_TPM,
alias=SAMPLES_ALIAS,
samples=SAMPLES,
group=GROUP,
treat="{treat}",
ctrl="{ctrl}",
log2fc=LOG2FC,
padj=FDR
script:
"scripts/annotateDEGs.py"
rule pheatmap_degs:
input:
degstab="differential_expression/diff_{treat}_vs_{ctrl}/diff_{treat}_vs_{ctrl}_results.txt",
image="quant/deseq2.ntd.RData"
output:
"differential_expression/diff_{treat}_vs_{ctrl}/diff_{treat}_vs_{ctrl}_all.degs.pdf",
"differential_expression/diff_{treat}_vs_{ctrl}/diff_{treat}_vs_{ctrl}_top20genes.pdf"
params:
treat="{treat}",
ctrl="{ctrl}",
padj=FDR,
topgene=20,
script:
"scripts/pheatmapDEGs.R"
rule anno_samples:
input:
GTF_Genes,
GTF_Trans,
"quant/gene_expression.TPM.txt",
"quant/transcripts_expression.TPM.txt",
output:
"quant/gene_expression.TPM.annotated.csv",
"quant/transcripts_expression.TPM.annotated.csv",
params:
group=GROUP,
alias=SAMPLES_ALIAS,
samples=SAMPLES,
script:
"scripts/annotateTPMs.py"
rule GSEA_Enrichr:
input:
"differential_expression/diff_{treat}_vs_{ctrl}/diff_{treat}_vs_{ctrl}_results.annotated.xls"
output:
GSEA_RES,
#directory("differential_expression/GO/GSEA_{treat}_vs_{ctrl}",
directory("differential_expression/GO/Enrichr_{treat}_vs_{ctrl}")
params:
treat="{treat}",
ctrl="{ctrl}",
log2fc=LOG2FC,
padj=FDR,
go=GO_DOMAIN,
script:
"scripts/gseaEnrichr.py"