New CLP to trim homopolymer starting sequence

src/java/org/broadinstitute/dropseqrna/barnyard/digitalallelecounts/SequenceBaseEnum.java

@@ -37,7 +37,7 @@ public enum SequenceBaseEnum {
 	T('T'),
 	N('N');
 
-	private final char base;
+	public final char base;
 
 	SequenceBaseEnum(char b) {
 		this.base=b;

src/java/org/broadinstitute/dropseqrna/readtrimming/AbstractTrimmerClp.java

@@ -27,6 +27,7 @@
 import htsjdk.samtools.util.CollectionUtil;
 import htsjdk.samtools.util.Histogram;
 import org.broadinstitute.barclay.argparser.Argument;
+import org.broadinstitute.dropseqrna.cmdline.CustomCommandLineValidationHelper;
 import picard.cmdline.CommandLineProgram;
 import picard.cmdline.StandardOptionDefinitions;
 
@@ -49,7 +50,7 @@ public abstract class AbstractTrimmerClp extends CommandLineProgram {
     public File OUTPUT_SUMMARY;
     @Argument(doc = "Which reads to trim.  0: unpaired reads; 1: first of pair; 2: second of pair")
     public List<Integer> WHICH_READ = new ArrayList<>(Arrays.asList(0));
-    protected Integer readsTrimmed = 0;
+    protected int readsTrimmed = 0;
     protected int numReadsTotal = 0;
     protected final Histogram<Integer> numBasesTrimmed = new Histogram<>();
 
@@ -65,4 +66,16 @@ protected boolean shouldTrim(final SAMRecord r) {
         }
         return false;
     }
+
+    @Override
+    protected String[] customCommandLineValidation() {
+        final ArrayList<String> list = new ArrayList<>(1);
+        if (!VALID_WHICH_READ.containsAll(WHICH_READ)) {
+            list.add("WHICH_READ must be one of " + VALID_WHICH_READ);
+        }
+        if (WHICH_READ.isEmpty()) {
+            list.add("WHICH_READ must be specified");
+        }
+        return CustomCommandLineValidationHelper.makeValue(super.customCommandLineValidation(), list);
+    }
 }

src/java/org/broadinstitute/dropseqrna/readtrimming/TrimHomopolymerStartingSequence.java

@@ -0,0 +1,172 @@
+/*
+ * MIT License
+ *
+ * Copyright 2025 Broad Institute
+ *
+ * Permission is hereby granted, free of charge, to any person obtaining a copy
+ * of this software and associated documentation files (the "Software"), to deal
+ * in the Software without restriction, including without limitation the rights
+ * to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
+ * copies of the Software, and to permit persons to whom the Software is
+ * furnished to do so, subject to the following conditions:
+ *
+ * The above copyright notice and this permission notice shall be included in all
+ * copies or substantial portions of the Software.
+ *
+ * THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
+ * IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
+ * FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
+ * AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
+ * LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
+ * OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE
+ * SOFTWARE.
+ */
+package org.broadinstitute.dropseqrna.readtrimming;
+
+import htsjdk.samtools.*;
+import htsjdk.samtools.metrics.MetricBase;
+import htsjdk.samtools.metrics.MetricsFile;
+import htsjdk.samtools.util.*;
+import org.broadinstitute.barclay.argparser.Argument;
+import org.broadinstitute.barclay.argparser.CommandLineProgramProperties;
+import org.broadinstitute.dropseqrna.barnyard.digitalallelecounts.SequenceBaseEnum;
+import org.broadinstitute.dropseqrna.cmdline.CustomCommandLineValidationHelper;
+import org.broadinstitute.dropseqrna.cmdline.DropSeq;
+import org.broadinstitute.dropseqrna.utils.SamHeaderUtil;
+
+import java.util.ArrayList;
+import java.util.Arrays;
+
+@CommandLineProgramProperties(summary = "Trim homopolymer run from beginning of reads.  If the entire read" +
+        " is a homopolymer run, instead of trimming all bases are set to Q3.",
+        oneLineSummary = "Trim the given sequence from the beginning of reads",
+        programGroup = DropSeq.class)
+public class TrimHomopolymerStartingSequence
+extends AbstractTrimmerClp {
+    public static final byte FULL_TRIM_QUALITY_SCORE = 3;
+    private final Log log = Log.getInstance(TrimHomopolymerStartingSequence.class);
+    private int readsCompletelyTrimmed = 0;
+
+    @Argument(doc = "How many mismatches are acceptable in the homopolymer run.")
+    public int MISMATCHES = 1;
+
+    @Argument(doc = "How many bases of homopolymer qualifies as a run to be trimmed.")
+    public int NUM_BASES = 6;
+
+    @Argument(doc = "The base to trim.")
+    public SequenceBaseEnum BASE = SequenceBaseEnum.T;
+    private byte BASE_BYTE;
+
+    @Override
+    protected String[] customCommandLineValidation() {
+        final ArrayList<String> list = new ArrayList<>(1);
+        if (NUM_BASES < 1) {
+            list.add("NUM_BASES must be greater than 0.");
+        }
+        if (MISMATCHES < 0) {
+            list.add("MISMATCHES must be greater than or equal to 0.");
+        }
+        return CustomCommandLineValidationHelper.makeValue(super.customCommandLineValidation(), list);
+    }
+
+    @Override
+    protected int doWork() {
+        IOUtil.assertFileIsReadable(INPUT);
+        IOUtil.assertFileIsWritable(OUTPUT);
+        BASE_BYTE = StringUtil.charToByte(BASE.base);
+        if (!SequenceUtil.isValidBase(BASE_BYTE)) {
+            throw new IllegalArgumentException(String.format("BASE (%c) must be a valid base, one of ACGT.", BASE));
+        }
+        final ProgressLogger progress = new ProgressLogger(log);
+
+        SamReader bamReader = SamReaderFactory.makeDefault().open(this.INPUT);
+        SAMFileHeader header = bamReader.getFileHeader();
+        SamHeaderUtil.addPgRecord(header, this);
+        SAMFileWriter writer= new SAMFileWriterFactory().makeSAMOrBAMWriter(header, true, OUTPUT);
+        for (SAMRecord r : bamReader) {
+            if (shouldTrim(r)) {
+                maybeClip(r);
+            }
+            writer.addAlignment(r);
+            progress.record(r);
+            this.numReadsTotal++;
+        }
+
+        CloserUtil.close(bamReader);
+
+        writer.close();
+        log.info("Number of reads trimmed: " + this.readsTrimmed, " total reads: " + this.numReadsTotal);
+        if (this.OUTPUT_SUMMARY!=null) writeSummary();
+
+        return 0;
+    }
+
+    private void maybeClip(final SAMRecord r) {
+        final byte[] readBases = r.getReadBases();
+        final int trimPosition = getTrimPosition(readBases);
+        if (trimPosition > 0) {
+            if (trimPosition == readBases.length) {
+                // if the entire read is a homopolymer run, set all qualities to Q3
+                byte[] value = new byte[readBases.length];
+                Arrays.fill(value, FULL_TRIM_QUALITY_SCORE);
+                r.setBaseQualities(value);
+                this.readsCompletelyTrimmed++;
+            } else {
+                final byte[] newReadBases = new byte[readBases.length - trimPosition];
+                System.arraycopy(readBases, trimPosition, newReadBases, 0, newReadBases.length);
+                r.setReadBases(newReadBases);
+                final byte[] readQuals = r.getBaseQualities();
+                final byte[] newReadQuals = new byte[readQuals.length - trimPosition];
+                System.arraycopy(readQuals, trimPosition, newReadQuals, 0, newReadQuals.length);
+                r.setBaseQualities(newReadQuals);
+            }
+            this.readsTrimmed++;
+        }
+        this.numBasesTrimmed.increment(trimPosition);
+    }
+
+    private int getTrimPosition(final byte[] readBases) {
+        if (readBases.length < NUM_BASES) {
+            return 0;
+        }
+        int trimPosition = readBases.length;
+        int numMismatches = 0;
+        for (int i = 0; i < readBases.length; i++) {
+            if (!SequenceUtil.basesEqual(readBases[i], BASE_BYTE)) {
+                if (++numMismatches > MISMATCHES) {
+                    trimPosition = i;
+                    break;
+                }
+            }
+        }
+        if (trimPosition < NUM_BASES) {
+            return 0;
+        }
+        if (numMismatches == trimPosition) {
+            // handle case in which all that was seen were mismatches.  This shouldn't happen unless MISMATCHES > 1
+            return 0;
+        }
+        return trimPosition;
+    }
+
+    private void writeSummary() {
+        final TrimMetric metric = new TrimMetric();
+        metric.TRIMMED_READS = this.readsTrimmed;
+        metric.TOTAL_READS = this.numReadsTotal;
+        metric.COMPLETED_TRIMMED_READS = this.readsCompletelyTrimmed;
+        MetricsFile<TrimMetric, Integer> mf = new MetricsFile<>();
+        mf.addMetric(metric);
+        mf.addHistogram(this.numBasesTrimmed);
+        mf.write(this.OUTPUT_SUMMARY);
+    }
+
+    public static class TrimMetric extends MetricBase {
+        public long TRIMMED_READS = 0;
+        public long TOTAL_READS = 0;
+        public long COMPLETED_TRIMMED_READS = 0;
+    }
+
+    public static void main(String[] args) {
+        new TrimHomopolymerStartingSequence().instanceMainWithExit(args);
+    }
+}

src/java/org/broadinstitute/dropseqrna/readtrimming/TrimStartingSequence.java

@@ -135,12 +135,6 @@ protected String[] customCommandLineValidation() {
 		if (MISMATCH_RATE != null && (MISMATCH_RATE < 0 || MISMATCH_RATE >= 1)) {
 			list.add("MISMATCH_RATE must be >= 0 and < 1");
 		}
-		if (!VALID_WHICH_READ.containsAll(WHICH_READ)) {
-			list.add("WHICH_READ must be one of " + VALID_WHICH_READ);
-		}
-		if (WHICH_READ.isEmpty()) {
-			list.add("WHICH_READ must be specified");
-		}
 		return CustomCommandLineValidationHelper.makeValue(super.customCommandLineValidation(), list);
 	}
 

src/tests/java/org/broadinstitute/dropseqrna/readtrimming/ClipReadsTest.java

@@ -46,7 +46,6 @@ public void testClipReads() {
         final ClipReads clp = new ClipReads();
         clp.INPUT = PAIRED_INPUT;
         clp.OUTPUT = TestUtils.getTempReportFile("ClipReadsTest.", ".sam");
-        clp.OUTPUT.deleteOnExit();
         clp.TMP_DIR = Collections.singletonList(tempDir);
         clp.BASE_RANGE = "1-16:17-28";
         clp.WHICH_READ = Collections.singletonList(AbstractTrimmerClp.FIRST_OF_PAIR);

src/tests/java/org/broadinstitute/dropseqrna/readtrimming/TrimHomopolymerStartingSequenceTest.java

@@ -0,0 +1,91 @@
+/*
+ * MIT License
+ *
+ * Copyright 2025 Broad Institute
+ *
+ * Permission is hereby granted, free of charge, to any person obtaining a copy
+ * of this software and associated documentation files (the "Software"), to deal
+ * in the Software without restriction, including without limitation the rights
+ * to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
+ * copies of the Software, and to permit persons to whom the Software is
+ * furnished to do so, subject to the following conditions:
+ *
+ * The above copyright notice and this permission notice shall be included in all
+ * copies or substantial portions of the Software.
+ *
+ * THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
+ * IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
+ * FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
+ * AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
+ * LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
+ * OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE
+ * SOFTWARE.
+ */
+package org.broadinstitute.dropseqrna.readtrimming;
+
+import htsjdk.samtools.SAMRecord;
+import htsjdk.samtools.SAMRecordIterator;
+import htsjdk.samtools.SamReader;
+import htsjdk.samtools.SamReaderFactory;
+import htsjdk.samtools.util.CloserUtil;
+import org.broadinstitute.dropseqrna.utils.TestUtils;
+import org.testng.Assert;
+import org.testng.annotations.Test;
+
+import java.io.File;
+import java.util.Arrays;
+import java.util.Collections;
+
+public class TrimHomopolymerStartingSequenceTest {
+    private static final File TESTDATA_DIR = new File("testdata/org/broadinstitute/dropseq/readtrimming");
+    private static final File INPUT = new File(TESTDATA_DIR, "prePolyTTrim.paired.sam");
+
+    @Test
+    public void testBasic() {
+        final File tempDir = TestUtils.createTempDirectory("TrimHomopolymerStartingSequenceTest.");
+        final TrimHomopolymerStartingSequence clp = new TrimHomopolymerStartingSequence();
+        clp.INPUT = INPUT;
+        clp.OUTPUT = TestUtils.getTempReportFile("TrimHomopolymerStartingSequenceTest.", ".sam");
+        clp.WHICH_READ = Collections.singletonList(AbstractTrimmerClp.FIRST_OF_PAIR);
+        clp.TMP_DIR = Collections.singletonList(tempDir);
+        Assert.assertEquals(clp.doWork(), 0);
+
+        final SamReader inputReader = SamReaderFactory.makeDefault().open(clp.INPUT);
+        final SamReader actualReader = SamReaderFactory.makeDefault().open(clp.OUTPUT);
+        final SAMRecordIterator inputIterator = inputReader.iterator();
+        final SAMRecordIterator actualIterator = actualReader.iterator();
+        while (inputIterator.hasNext() && actualIterator.hasNext()) {
+            final SAMRecord inputRecord = inputIterator.next();
+            final SAMRecord actualRecord = actualIterator.next();
+            final String readName = actualRecord.getReadName();
+            Assert.assertEquals(readName, inputRecord.getReadName());
+            Assert.assertEquals(actualRecord.getFirstOfPairFlag(), inputRecord.getFirstOfPairFlag(), readName);
+            if (actualRecord.getFirstOfPairFlag()) {
+                final String[] readNameFields = readName.split(":");
+                if (readNameFields[0].equals("trimmed")) {
+                    final int trimLength = Integer.parseInt(readNameFields[1]);
+                    final String inputRead = inputRecord.getReadString().substring(trimLength);
+                    Assert.assertEquals(actualRecord.getReadString(), inputRead, readName);
+                    final String inputQual = inputRecord.getBaseQualityString().substring(trimLength);
+                    Assert.assertEquals(actualRecord.getBaseQualityString(), inputQual, readName);
+                } else if (readNameFields[0].equals("notrim")) {
+                    Assert.assertEquals(actualRecord, inputRecord);
+                } else if (readNameFields[0].equals("fulltrim")) {
+                    Assert.assertEquals(actualRecord.getReadString(), inputRecord.getReadString(), readName);
+                    byte[] expectedQuals = new byte[inputRecord.getReadLength()];
+                    Arrays.fill(expectedQuals, TrimHomopolymerStartingSequence.FULL_TRIM_QUALITY_SCORE);
+                    Assert.assertEquals(actualRecord.getBaseQualities(), expectedQuals, readName);
+                } else {
+                    Assert.fail("Unexpected read name: " + readName);
+                }
+            } else {
+                Assert.assertEquals(actualRecord, inputRecord);
+            }
+        }
+        Assert.assertFalse(inputIterator.hasNext());
+        Assert.assertFalse(actualIterator.hasNext());
+        CloserUtil.close(inputReader);
+        CloserUtil.close(actualReader);
+
+    }
+}

testdata/org/broadinstitute/dropseq/readtrimming/paired_end.28_technical.sam

@@ -1,8 +1,8 @@
 @HD	VN:1.6	GO:none	SO:queryname
-@RG	ID:A	SM:v29_10X-GEMX-3P_ic_rxn1	LB:v29_10X-GEMX-3P_ic_rxn1	PL:ILLUMINA	PU:22FKMMLT4.ACCAGACAAC.CCTAGTTCCT.1	CN:BI
-LH00453:184:22FKMMLT4:1:1101:10031:9853	77	*	0	0	*	*	0	0	ACCGGCTGTGAGACTACGCTGCGCAGCCTTGTTTTTTTTTTTTTTTTTTTTTCTGACCGTCTTCGAACCTCCCCCCTTCGTTCTTGCTTAATGCCAACATTCTTCGCAAATGCTTTCTATCTTATCCTTCTTTTACCCACCCAAAAACCT	-I9II9IIIIII9IIIIIII9IIIIII-9-------9I9999999I9I99-----99--9-99--99---------99--9--99--99-9999-99-9---99--99999-999---------99I--99---999---9---9----9	XC:Z:ACCGGCTGTGAGACTA	RG:Z:A
-LH00453:184:22FKMMLT4:1:1101:10031:9853	141	*	0	0	*	*	0	0	GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG	I--I----99II9I9I-I-9--I9I99999-I--99I----9-99------------9-99-9---9--99--9999-99-9-99----999-9--999--9-9999-9--999999-I99-I9-9I99I99---9-9999999-9999-	XC:Z:ACCGGCTGTGAGACTA	RG:Z:A	XM:Z:CGCTGCGCAGCC
-LH00453:184:22FKMMLT4:1:1101:31945:11394	77	*	0	0	*	*	0	0	ATGCTAGTCACCCTGCTCTGCACTAGACTTTTTTTTTTTTTTTTTTTTTTGTAGTCACCAAAACTGTGAGGGGAGAAACCCCAAGCTCTCCCGCAAACCTCTCTCTCTCGTCACAACTATGTCTATTCCCCTTAACTTTTTCAAACAACC	IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII---9--9I-I99I9----99I99--99I9---99-99-99--9-9-9--9-----9I--------9-9-9---9-----9------9------9------	XC:Z:ATGCTAGTCACCCTGC	RG:Z:A
-LH00453:184:22FKMMLT4:1:1101:31945:11394	141	*	0	0	*	*	0	0	AGAATGAAGGCAATCAATATTTTTCACTTGCTTCTCCACTAAGTTTGTTATTGCACCCATTATTCAAACTTTCAAATTCCTGGTTGAAAAAATGCTCATCTTCAAAATTTACTATTAACAGCCACAATTGTCACCATATATATATATTTG	IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII	XC:Z:ATGCTAGTCACCCTGC	RG:Z:A	XM:Z:TCTGCACTAGAC
-LH00453:184:22FKMMLT4:1:1101:6236:13425	77	*	0	0	*	*	0	0	CTGTCATTCAGGAACGTGTGTGCCCAGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGACCAAGAAACCCCCCACCCAACAAAAAAAAACCAACACCCACAAACACTTTTCCGCTTTAAAACACCCACAACAAAAAATTTTTTTCCCAAG	IIIII9IIIIII-IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIII-I--I9-99I-9I-I-9---I-I9--I9I-9I9---99-9-9---999II--9I9-9---9-9---------99--9--I9--9-9---9-----	XC:Z:CTGTCATTCAGGAACG	RG:Z:A
-LH00453:184:22FKMMLT4:1:1101:6236:13425	141	*	0	0	*	*	0	0	CAATGGTGCCTGTCCAGATTTTCTGACAGTCACCTCAGGAAATTGACACAGACACGCTGTGATCCTGGTTTCTGTACAGTTACTTTGTGTGTTACTCAGCACCTGGGCTGTTAAGGGTATTATCAAGCGTGAAAAAATAAAGACAAAAAC	IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII	XC:Z:CTGTCATTCAGGAACG	RG:Z:A	XM:Z:TGTGTGCCCAGC
+@RG	ID:A	SM:rxn1	LB:rxn1	PL:ILLUMINA	PU:123456789.ACCAGACAAC.CCTAGTTCCT.1	CN:BI
+LH00453:184:123456789:1:1101:10031:9853	77	*	0	0	*	*	0	0	ACCGGCTGTGAGACTACGCTGCGCAGCCTTGTTTTTTTTTTTTTTTTTTTTTCTGACCGTCTTCGAACCTCCCCCCTTCGTTCTTGCTTAATGCCAACATTCTTCGCAAATGCTTTCTATCTTATCCTTCTTTTACCCACCCAAAAACCT	-I9II9IIIIII9IIIIIII9IIIIII-9-------9I9999999I9I99-----99--9-99--99---------99--9--99--99-9999-99-9---99--99999-999---------99I--99---999---9---9----9	XC:Z:ACCGGCTGTGAGACTA	RG:Z:A
+LH00453:184:123456789:1:1101:10031:9853	141	*	0	0	*	*	0	0	GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG	I--I----99II9I9I-I-9--I9I99999-I--99I----9-99------------9-99-9---9--99--9999-99-9-99----999-9--999--9-9999-9--999999-I99-I9-9I99I99---9-9999999-9999-	XC:Z:ACCGGCTGTGAGACTA	RG:Z:A	XM:Z:CGCTGCGCAGCC
+LH00453:184:123456789:1:1101:31945:11394	77	*	0	0	*	*	0	0	ATGCTAGTCACCCTGCTCTGCACTAGACTTTTTTTTTTTTTTTTTTTTTTGTAGTCACCAAAACTGTGAGGGGAGAAACCCCAAGCTCTCCCGCAAACCTCTCTCTCTCGTCACAACTATGTCTATTCCCCTTAACTTTTTCAAACAACC	IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII---9--9I-I99I9----99I99--99I9---99-99-99--9-9-9--9-----9I--------9-9-9---9-----9------9------9------	XC:Z:ATGCTAGTCACCCTGC	RG:Z:A
+LH00453:184:123456789:1:1101:31945:11394	141	*	0	0	*	*	0	0	AGAATGAAGGCAATCAATATTTTTCACTTGCTTCTCCACTAAGTTTGTTATTGCACCCATTATTCAAACTTTCAAATTCCTGGTTGAAAAAATGCTCATCTTCAAAATTTACTATTAACAGCCACAATTGTCACCATATATATATATTTG	IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII	XC:Z:ATGCTAGTCACCCTGC	RG:Z:A	XM:Z:TCTGCACTAGAC
+LH00453:184:123456789:1:1101:6236:13425	77	*	0	0	*	*	0	0	CTGTCATTCAGGAACGTGTGTGCCCAGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGACCAAGAAACCCCCCACCCAACAAAAAAAAACCAACACCCACAAACACTTTTCCGCTTTAAAACACCCACAACAAAAAATTTTTTTCCCAAG	IIIII9IIIIII-IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIII-I--I9-99I-9I-I-9---I-I9--I9I-9I9---99-9-9---999II--9I9-9---9-9---------99--9--I9--9-9---9-----	XC:Z:CTGTCATTCAGGAACG	RG:Z:A
+LH00453:184:123456789:1:1101:6236:13425	141	*	0	0	*	*	0	0	CAATGGTGCCTGTCCAGATTTTCTGACAGTCACCTCAGGAAATTGACACAGACACGCTGTGATCCTGGTTTCTGTACAGTTACTTTGTGTGTTACTCAGCACCTGGGCTGTTAAGGGTATTATCAAGCGTGAAAAAATAAAGACAAAAAC	IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII	XC:Z:CTGTCATTCAGGAACG	RG:Z:A	XM:Z:TGTGTGCCCAGC