hartleys
commented
4 years ago So a few things:
It looks like you have a big complicated expression to get the RG entry? Are you sure that thing is working properly? Have you tried running it separately? Have you tried running it with the RG stated explicitly? Also your expressions for RG and fastq don't appear to be inside a $(), so wouldn't they just be interpreted as text? I'm not sure how this is running at all? What shell are you using?
Also, the error itself seems to be that the RG field doesn't have an SM tag, so it is trying to extract the sample name and failing I guess? It looks like this is a restriction built into the HTSeq library, so I can't fix it. The problem might be solved by adding an SM tag to each RG entry. See the SAM spec: https://samtools.github.io/hts-specs/SAMv1.pdf (which it looks like you're already pretty familiar with).
Dear Doctor, I am running the code on my bam files and I get FATAL error message. here below my code: for i in $(ls $RDS/projects/seqdataset/ephemeral/RNASEQ/RNAseqROX/140725_0281_IP/140725_SN674_0281_AC47BHACXX/Unaligned/Project_IP) do java -Xmx4G -jar $RDS/home/anaconda3/pkgs/qorts-1.3.6-0/share/qorts-1.3.6-0/QoRTs.jar QC \ --generatePlots \ --addFunctions mismatchEngine,annotatedSpliceExonCounts,FPKM,writeGeneBodyIv,fastqUtils,writeDocs,makeJunctionBed,makeWiggles,makeAllBrowserTracks,calcDetailedGeneCounts \ --verbose \ --stranded \ --readGroup
ls $RDS/projects/seqdataset/ephemeral/RNASEQ/RNAseqROX/140725_0281_IP/140725_SN674_0281_AC47BHACXX/Unaligned/Project_IP/$i/*_R1_*.fastq.gz | sort | awk '{gsub(/.*\//,""); gsub(/.fastq.gz/,""); gsub(/R1/,""); printf "ID:" $i " , "}' | head -c -3\ --outfilePrefix $i \ --chromSizes $RDS/projects/sequ/live/Genecode/mouseindex/chrNameLength.txt \ --rawfastqls $RDS/projects/seqdataset/ephemeral/RNASEQ/RNAseqROX/140725_0281_IP/140725_SN674_0281_AC47BHACXX/Unaligned/Project_IP/$i/*.fastq.gz | sort | tr "\n" ","\ --genomeFA $RDS/projects/sequ/live/Genecode/mouseannotation/GRCm38.primaryassembly.genome.fa \ $RDS/projects/sequ/live/mapped/Genecodesecondpassfilter/ROX$i*.sortedByCoord.out.bam \ $RDS/projects/sequ/live/Genecode/mouseannotation/gencode.vM25.primary_assembly.annotation.gtf \ $RDS/projects/sequ/live/mapped/QoRT_QC/$i doneand the error message is:
oRTs encountered a FATAL ERROR. For general help, use command: java -jar path/to/jar/QoRTs.jar --man ============================FATAL_ERROR============================ Error info: Exception in thread "main" net.sf.samtools.SAMFormatException: Error parsing SAM header. @RG line missing SM tag. Line: @RG ID:SNA2_TGACCA_L008__001; File /rds/general/user/ipalmisa/projects/sequ/live/mapped/Genecodesecondpassfilter/ROX_Sample_SNA2Aligned.sortedByCoord.out.bam; Line number 69 at net.sf.samtools.SAMTextHeaderCodec.reportErrorParsingLine(SAMTextHeaderCodec.java:234) at net.sf.samtools.SAMTextHeaderCodec.access$200(SAMTextHeaderCodec.java:40) at net.sf.samtools.SAMTextHeaderCodec$ParsedHeaderLine.requireTag(SAMTextHeaderCodec.java:316) at net.sf.samtools.SAMTextHeaderCodec.parseRGLine(SAMTextHeaderCodec.java:164) at net.sf.samtools.SAMTextHeaderCodec.decode(SAMTextHeaderCodec.java:97) at net.sf.samtools.BAMFileReader.readHeader(BAMFileReader.java:492) at net.sf.samtools.BAMFileReader.(BAMFileReader.java:162)
at net.sf.samtools.BAMFileReader.(BAMFileReader.java:121)
at net.sf.samtools.SAMFileReader.init(SAMFileReader.java:647)
at net.sf.samtools.SAMFileReader.(SAMFileReader.java:184)
at net.sf.samtools.SAMFileReader.(SAMFileReader.java:139)
at qcUtils.runAllQC$.runOnSeqFile(runAllQC.scala:1023)
at qcUtils.runAllQC$.run(runAllQC.scala:970)
at qcUtils.runAllQC$allQC_runner.run(runAllQC.scala:680)
at runner.runner$.main(runner.scala:98)
at runner.runner.main(runner.scala)
Can you please help me in this? thanks BW ilaria