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Following Best practice, I have ERROR:MATE_NOT_FOUND in all my files

I am running Mutect2 on about 100 pairs of tumor-normal samples of whole exome sequencing. I am using GATK4.0.9.0. The pipeline used the recommended best practice pipeline:
1. FastqToSam,
2. MarkIlluminaAdapters,
3. SamToFastq, bwa (mem -M -t4),
4. MergeBamAlignment (CREATE_INDEX=true, ADD_MATE_CIGAR=true, CLIP_ADAPTERS=false, CLIP_OVERLAPPING_READS=true, INCLUDE_SECONDARY_ALIGNMENTS=true, MAX_INSERTIONS_OR_DELETIONS=-1, PRIMARY_ALIGNMENT_STRATEGY=MostDistant, ATTRIBUTES_TO_RETAIN=XS )
5. MarkDuplicates (VALIDATION_STRINGENCY SILENT, OPTICAL_DUPLICATE_PIXEL_DISTANCE=2500, CREATE_INDEX=true)
6. BaseRecalibrator (using 100bp-padded interval bed file of the exome design)
7. ApplyBQSR
8. generate the PON VCF successfully using all normal samples.
9. When I tried to run Mutect2 to call the somatic short variants, all pairs went through the analysis except one pair which failed to pass the same region after several trials. This part of the log after one failed trail:
...
19:12:18.761 INFO ProgressMeter - chr1:41708411 5.8 5020 858.5
19:12:28.648 INFO PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 262.425723624
19:12:28.649 INFO SmithWatermanAligner - Total compute time in java Smith-Waterman : 13.26 sec
19:12:29.035 INFO Mutect2 - Shutting down engine
[October 10, 2018 7:12:29 PM EDT] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 6.09 minutes.
Runtime.totalMemory()=2715287552
java.lang.IllegalArgumentException: readMaxLength must be > 0 but got 0
...

We I tested the BAM files using ValidateSamFile, I got these results for tumor and normal samples:
Error Type Count
ERROR:MATE_NOT_FOUND 3622058

Error Type Count
ERROR:MATE_NOT_FOUND 2773161

I used the FixMateInformation tool to fix the files, re-indexed, and re-ran Mutect2 successfully but I tested all BAM files and found them carrying the same error! Why did this happen? is it a bug or did I do something wrong? Why did one pair fail to go through the variant calling step while all other pairs did not? Does this affect the quality of variant calls?

Thank you

Best Answer

Answers

  • davidbendavidben BostonMember, Broadie, Dev ✭✭

    Just to complement what @bhanuGandham said:

    Why did one pair fail to go through the variant calling step while all other pairs did not?

    Most likely the other samples' affected reads did not occur near a potential somatic variant. In most of the genome M2 doesn't get as far as the code that threw the error. PairHMM is only reached if the initial pileup-based triage and local assembly indicate the existence of a somatic variant.

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