Failed to run bamqc net.sf.samtools.SAMFormatException: Error parsing SAM header. @RG line missing SM tag. Line: @RG

[Gabriel A. C. Gama]

Hello everyone! I've been trying to run qualimap bamqc and RNAseq QC with my BAM files

To make sure I have no problems on processing previously to qualimap, I used STAR aligner to output SAM files (sorted by name) using ensembl reference and gtf.

Then I converted the SAM files to BAM with samtools (-fixmate) then sorted it by coordinate with samtools also. Finally, I indexed these sorted bam files.

Then, I tried:

qualimap --java-mem-size=16G bamqc -bam /home/samtools/sorted_ensembl/101_FRAS202421991-1a_1.fqAligned.out-sorted.bam -gff /home/references/ENSEMBL/star/Homo_sapiens.GRCh38.103.chr_patch_hapl_scaff.gtf -gd HUMAN -ip -nt 4 -p non-strand-specific -outdir /home/qualimap/bamqc -outfile BAMQC_ensembl.pdf

Which gives me this error:

Selected tool: bamqc

Available memory (Mb): 32

Max memory (Mb): 15271

Sun Apr 25 14:08:37 GMT-03:00 2021              WARNING Output folder already exists, the results will be saved there

Starting bam qc....

Failed to run bamqc

net.sf.samtools.SAMFormatException: Error parsing SAM header. @RG line missing SM tag. Line:

@RG     ID:GRPundef; File /home/samtools/sorted_ensembl/101_FRAS202421991-1a_1.fqAligned.out-sorted.bam; Line number 199

        at net.sf.samtools.SAMTextHeaderCodec.reportErrorParsingLine(SAMTextHeaderCodec.java:230)

        at net.sf.samtools.SAMTextHeaderCodec.access$100(SAMTextHeaderCodec.java:39)

        at net.sf.samtools.SAMTextHeaderCodec$ParsedHeaderLine.requireTag(SAMTextHeaderCodec.java:306)

        at net.sf.samtools.SAMTextHeaderCodec.parseRGLine(SAMTextHeaderCodec.java:160)

        at net.sf.samtools.SAMTextHeaderCodec.decode(SAMTextHeaderCodec.java:93)

        at net.sf.samtools.BAMFileReader.readHeader(BAMFileReader.java:393)

        at net.sf.samtools.BAMFileReader.<init>(BAMFileReader.java:146)

        at net.sf.samtools.BAMFileReader.<init>(BAMFileReader.java:114)

        at net.sf.samtools.SAMFileReader.init(SAMFileReader.java:514)

        at net.sf.samtools.SAMFileReader.<init>(SAMFileReader.java:167)

        at net.sf.samtools.SAMFileReader.<init>(SAMFileReader.java:122)

        at org.bioinfo.ngs.qc.qualimap.process.BamStatsAnalysis.run(BamStatsAnalysis.java:202)

        at org.bioinfo.ngs.qc.qualimap.main.BamQcTool.execute(BamQcTool.java:242)

        at org.bioinfo.ngs.qc.qualimap.main.NgsSmartTool.run(NgsSmartTool.java:190)

        at org.bioinfo.ngs.qc.qualimap.main.NgsSmartMain.main(NgsSmartMain.java:111)

I've observed the same error for all samples, in the same line

[Konstantin Okonechnikov]

Hi!

Seems to be the issue with header parsing. Could you perhaps share a small subsample from this BAM file (http://okko73313.blogspot.com/2013/03/random-subsample-from-bam-file.html)? Or at least the header to check it in detail.

Best regards,

   Konstantin

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[Gabriel Gama]

Hello Konstantin!

I transferred 5% of reads from one of the files:

https://transfer.sh/Fn94n/101_random.bam

To view this discussion on the web visit https://groups.google.com/d/msgid/qualimap/CAMe83jniz3CdFW2F7cD0wAjWrKg9%3DXBcMA9VErmnFmFvcypbww%40mail.gmail.com.

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Atenciosamente,

Gabriel A. C. Gama
Universidade Federal de São Paulo - UNIFESP
Laboratório Bases Genéticas dos Tumores da Tiroide
Rua Pedro de Toledo, 669 - 11º andar Vila Clementino São Paulo/SP - 04039-032
(11) 5576-4979 / (11) 999731226

[Konstantin Okonechnikov]

The issue was due to incorrectly formatted according to SAM format read group - the SM tag is missing: 

@RG     ID:GRPundef 

This happened due to dependence on picard library requirements for SAM parser. Simply removing it from header fixes the situation, since Qualimap does not require read group in the analysis. Something like this:

samtools view -h 101_random.bam | grep -v "^@RG" > 101_random.sam

Possible solution is to clean header and save to temporary BAM file, run Qualimap and then remove the file or fix the read group in the BAM file. 

Best regards,

   Konstantin

To view this discussion on the web visit https://groups.google.com/d/msgid/qualimap/CAMjxR3CmVmjLA0T945LpKeTobwnG5inn3hpV%3Du36q4%3DDemZcZw%40mail.gmail.com.

[Gabriel A. C. Gama]

This worked, and I got BAMqc to run just fine.