Unable to vizualise bam files

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constan...@gmail.com

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Nov 30, 2018, 5:17:50 AM11/30/18

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Hi everybody,

I'm trying to visualize some bam files with IGV, but it's not working... I have seen that other people had this kind of problem but I couldn't solve it.

Here is a capture. Chromosome names seem similar in the sequence file and in the bam file

Here is how the bam file looks when using samtools:

samtools1.2 view LNMR121.pos.bam |head

K00201:257:H27N7BBXY:4:2223:26575:8576 272 chr1 235 0 50M * 0 0 AAGACAGAATCAGGCATGAGGAGTTCATGGCGATGAGAGTGAGTGACAAG JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA NH:i:5 HI:i:5 NM:i:0 MD:Z:50 AS:i:49 nM:i:0 jM:B:c,-1 jI:B:i,-1

K00201:257:H27N7BBXY:4:1107:32502:7697 16 chr1 285 3 50M * 0 0 CCACTCGGGTCTCCTGTCTGAGAGTTGAGCTGCTTCCTCACAGATCTGCG JFJJJJJFJFJAAFFFJJFJFJJJFA<JJJJJAJJJJJFFJJJ7FF-AAA NH:i:2 HI:i:1 NM:i:0 MD:Z:50 AS:i:49 nM:i:0 jM:B:c,-1 jI:B:i,-1

K00201:257:H27N7BBXY:4:1123:30502:40385 272 chr1 713 0 1S49M * 0 0 AAGAGCTCTTTTTCTCCTCTCCTCTCTCAGTGCCCCTTCCTCTCTCCTAC J<AJJFFJJJJAJAF-7JJJJFAFJJFJJFJJJJJJJJJFJ<A<JFFFAA NH:i:10 HI:i:9 NM:i:0 MD:Z:49 AS:i:48 nM:i:0 jM:B:c,-1 jI:B:i,-1

Here when I look at the headers:

samtools1.2 view -H LNMR121.pos.bam

@HD VN:1.4 SO:coordinate

@SQ SN:chr1 LN:282763074

@SQ SN:chr10 LN:112626471

@SQ SN:chr11 LN:90463843

@SQ SN:chr12 LN:52716770

@SQ SN:chr13 LN:114033958

@SQ SN:chr14 LN:115493446

@SQ SN:chr15 LN:111246239

@SQ SN:chr16 LN:90668790

@SQ SN:chr17 LN:90843779

@SQ SN:chr18 LN:88201929

@SQ SN:chr19 LN:62275575

@SQ SN:chr2 LN:266435125

@SQ SN:chr20 LN:56205956

@SQ SN:chr3 LN:177699992

@SQ SN:chr4 LN:184226339

@SQ SN:chr5 LN:173707219

@SQ SN:chr6 LN:147991367

@SQ SN:chr7 LN:145729302

@SQ SN:chr8 LN:133307652

@SQ SN:chr9 LN:122095297

@SQ SN:chrM LN:16313

@SQ SN:chrX LN:159970021

@SQ SN:chrY LN:3310458

@PG ID:STAR PN:STAR VN:STAR_2.5.3a CL:STAR --runMode alignReads --runThreadN 12 --genomeDir /ifs/illumina/share/Utilities/Genomes/Rattus_norvegicus/rn6/STAR/Rattus_norvegicus.Rnor_6.0.92_UCSConlychr --readFilesIn /ifs/illumina/vagnec/Analyses/RNASeq_Rat/S18212_Rat/2018_10_08_pipeline/Spike/Alignment/bowtie2/LNMR121/LNMR121_nospike.fastq.gz --readFilesCommand zcat --outFileNamePrefix /ifs/illumina/vagnec/Analyses/RNASeq_Rat/S18212_Rat/2018_10_08_pipeline/Alignment/STAR/LNMR121/ --outReadsUnmapped Fastx --outSAMtype BAM SortedByCoordinate --outSAMattributes NH HI NM MD AS nM jM jI XS --sjdbGTFfile /ifs/illumina/share/Utilities/Genomes/Rattus_norvegicus/rn6/Annotations/Ensembl/Rattus_norvegicus.Rnor_6.0.92_UCSConlychr.gtf --quantMode GeneCounts --twopassMode Basic

@CO user command line: STAR --readFilesCommand zcat --quantMode GeneCounts --sjdbGTFfile /ifs/illumina/share/Utilities/Genomes/Rattus_norvegicus/rn6/Annotations/Ensembl/Rattus_norvegicus.Rnor_6.0.92_UCSConlychr.gtf --runThreadN 12 --outReadsUnmapped Fastx --outSAMattributes NH HI NM MD AS nM jM jI XS --outSAMtype BAM SortedByCoordinate --outFileNamePrefix /ifs/illumina/vagnec/Analyses/RNASeq_Rat/S18212_Rat/2018_10_08_pipeline/Alignment/STAR/LNMR121/ --readFilesIn /ifs/illumina/vagnec/Analyses/RNASeq_Rat/S18212_Rat/2018_10_08_pipeline/Spike/Alignment/bowtie2/LNMR121/LNMR121_nospike.fastq.gz --runMode alignReads --twopassMode Basic --genomeDir /ifs/illumina/share/Utilities/Genomes/Rattus_norvegicus/rn6/STAR/Rattus_norvegicus.Rnor_6.0.92_UCSConlychr

Thank you in advance for your help

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constan...@gmail.com

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Nov 30, 2018, 5:39:51 AM11/30/18

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Sorry, I forgot to add my IGV log:

INFO [2018-11-30T10:40:49,548] [Main.java:156] Startup IGV Version 2.4.16 11/17/2018 10:33 PM

INFO [2018-11-30T10:40:49,608] [Main.java:157] Java 1.8.0_191

INFO [2018-11-30T10:40:49,608] [Main.java:158] Default User Directory: C:\Users\vagnec\Documents

INFO [2018-11-30T10:40:49,608] [Main.java:159] OS: Windows 7

INFO [2018-11-30T10:40:53,064] [GenomeManager.java:186] Loading genome: C:\Users\vagnec\igv\genomes\rn6.genome

INFO [2018-11-30T10:40:56,281] [GenomeComboBox.java:79] Enter genome combo box

INFO [2018-11-30T10:40:56,306] [GenomeManager.java:277] Genome loaded. id= rn6

INFO [2018-11-30T10:40:56,333] [CommandListener.java:120] Listening on port 60151

INFO [2018-11-30T10:50:19,621] [IGV.java:1345] Loading 1 resources.

INFO [2018-11-30T10:50:19,634] [TrackLoader.java:122] Loading resource, path C:\Users\vagnec\Documents\S18212_Ins1\bam\LNMR121.pos.bam

ERROR [2018-11-30T10:50:25,983] [BAMReader.java:194] Error querying for sequence: chr20

java.lang.IllegalArgumentException: null

at java.nio.Buffer.position(Unknown Source) ~[?:1.8.0_191]

at htsjdk.samtools.AbstractBAMFileIndex$MemoryMappedFileBuffer.skipBytes(AbstractBAMFileIndex.java:508) ~[igv.jar:?]

at htsjdk.samtools.AbstractBAMFileIndex.skipBytes(AbstractBAMFileIndex.java:450) ~[igv.jar:?]

at htsjdk.samtools.AbstractBAMFileIndex.skipToSequence(AbstractBAMFileIndex.java:426) ~[igv.jar:?]

at htsjdk.samtools.AbstractBAMFileIndex.query(AbstractBAMFileIndex.java:277) ~[igv.jar:?]

at htsjdk.samtools.DiskBasedBAMFileIndex.getSpanOverlapping(DiskBasedBAMFileIndex.java:61) ~[igv.jar:?]

at htsjdk.samtools.BAMFileReader.getFileSpan(BAMFileReader.java:898) ~[igv.jar:?]

at htsjdk.samtools.BAMFileReader.createIndexIterator(BAMFileReader.java:915) ~[igv.jar:?]

at htsjdk.samtools.BAMFileReader.query(BAMFileReader.java:575) ~[igv.jar:?]

at htsjdk.samtools.SamReader$PrimitiveSamReaderToSamReaderAdapter.query(SamReader.java:528) ~[igv.jar:?]

at htsjdk.samtools.SamReader$PrimitiveSamReaderToSamReaderAdapter.query(SamReader.java:533) ~[igv.jar:?]

at org.broad.igv.sam.reader.BAMReader.query(BAMReader.java:191) ~[igv.jar:?]

at org.broad.igv.sam.AlignmentTileLoader.loadTile(AlignmentTileLoader.java:162) ~[igv.jar:?]

at org.broad.igv.sam.AlignmentDataManager.loadInterval(AlignmentDataManager.java:405) ~[igv.jar:?]

at org.broad.igv.sam.AlignmentDataManager.load(AlignmentDataManager.java:350) ~[igv.jar:?]

at org.broad.igv.sam.CoverageTrack.load(CoverageTrack.java:172) ~[igv.jar:?]

at org.broad.igv.ui.panel.DataPanel.lambda$load$3(DataPanel.java:225) ~[igv.jar:?]

at java.util.concurrent.CompletableFuture$AsyncRun.run(Unknown Source) [?:1.8.0_191]

at java.util.concurrent.ThreadPoolExecutor.runWorker(Unknown Source) [?:1.8.0_191]

at java.util.concurrent.ThreadPoolExecutor$Worker.run(Unknown Source) [?:1.8.0_191]

at java.lang.Thread.run(Unknown Source) [?:1.8.0_191]

INFO [2018-11-30T10:50:26,017] [AlignmentTrack.java:262] Experiment type = OTHER

ERROR [2018-11-30T10:50:26,023] [BAMReader.java:194] Error querying for sequence: chr20