I'm analyzing whole exam sequencing data, and using Picard to perform some QC on my aligned / sorted / duplicated removed .bam files. When running picard CollectSequencingArtifactMetrics, I receive the following error:
Exception in thread "main" picard.PicardException: Record contains library that is missing from header: UnknownLibrary
WES data processing / analysis is not my main area of expertise at all, and I'm not super familiar with the .sam / .bam formats. Does anyone have any idea what could be causing this? I've run other picard functions, such as SortSam and MarkDuplicates without errors, so I'm pretty confused.
This error happens when a read is not associated to a read group
1) did you declare the read group in the header @RG
2) do you have any read without the RG attribute ?
So I ran picard ValidateSamFile and did receive the error:
ERROR:MISSING_READ_GROUP
The .bam file was generated automatically as output from bwa mem, first in .sam format and then converted to .bam using picard SamFormatConverter. So, I'm guessing that I didn't declare the read group in the header (unless it's done automatically) and I'm not sure how to check if I have a read without the RG attribute.
you should/must have specified some read-groups. https://gatk.broadinstitute.org/hc/en-us/articles/360035890671-Read-groups
Specified them at what step? I ran samtools view -H | grep '^@RG' and got nothing in return, which I'm guessing means I failed to specify them at some point when I was supposed to.
EDIT: might this have something to do with how I combined the R[1-2]_L001.fastq & R[1-2]_L002.fastq files? I used:
zcat sample_R1_L001.fastq.gz sample_R1_L002.fastq.gz > sample_R1.fastq
to combine the lanes for both paired reads.
At the time of original alignment. Could add now: Adding Read Groups To Bam Files
Plain cat works for combining lane specific files.
it can be any string. You can use the sample name.
better/faster:
cat sample_R1_L001.fastq.gz sample_R1_L002.fastq.gz > sample_R1.fastq.gz
bwa can use gzipped fastq files
Furthermore, you can parallelize things by mapping each fastq and merge the bam later.
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Try
VALIDATION_STRINGENCY=LENIENTSame error, unfortunately.