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# Mapping, processing and duplicate marking with Picard tools: ValidateSamFile errors

Member

I am trying to follow the best practices for mapping my (Paired-end Illumina HiSeq) reads to the reference, by following this presentation:

From what I understand, I should use MergeBamAlignment to clean up the output from bwa, and then use this cleaned up output for the rest of the analysis. However, when I run ValidateSamFile after running MergeBamAlignment I get a lot of errors, and running CleanSam on the file does not resolve any of them. What am I doing wrong? I've tried searching the web for more details about MergeBamAlignment but I haven't been able to find much. Please let me know if you require any additional information.

How I ran MergeBamAlignment
picard-tools MergeBamAlignment \
REFERENCE_SEQUENCE=/path/to/reference.fasta \
PAIRED_RUN=true # Why is this needed?

Error report from ValidateSamFile
## HISTOGRAM java.lang.String
Error Type Count
ERROR:INVALID_CIGAR 5261
ERROR:MATES_ARE_SAME_END 30
ERROR:MISMATCH_FLAG_MATE_NEG_STRAND 30

Tagged:

Hi there,

Some of these errors aren't fixable by CleanSam. Try running FixMate to solve the mate pair errors. For the cigar errors, you'll be able to filter those out using -rf BadCigar in the subsequent GATK steps.

• Member

Thank you for your response. I've tried running FixMate on the file to fix the errors but evidently I'm doing something wrong, since this only leads to more errors!

picard-tools ValidateSamFile INPUT=input.bam MODE=SUMMARY

## HISTOGRAM    java.lang.String
Error Type  Count
ERROR:INVALID_CIGAR 5261
ERROR:MATES_ARE_SAME_END    30
ERROR:MISMATCH_FLAG_MATE_NEG_STRAND 30


Above is the sam validation for the original file. To fix these errors, I ran
picard-tools FixMateInformation INPUT=input.bam OUTPUT=output.bam VALIDATION_STRINGENCY=LENIENT

However, after running FixMateInformation the number of errors increased, and I'm not sure what I did wrong that could cause this.

picard-tools ValidateSamFile INPUT=output.bam MODE=SUMMARY

## HISTOGRAM    java.lang.String
Error Type  Count
ERROR:INVALID_CIGAR 5261
ERROR:MATES_ARE_SAME_END    27346
ERROR:MISMATCH_FLAG_MATE_NEG_STRAND 54
ERROR:MISMATCH_FLAG_MATE_UNMAPPED   108
`

Hi @Vanilla,

Sorry for the delayed response. I'm not 100% sure but I think this is expected. Some of these mapping issues can't be fixed as such, and if so, what the tools do is they flag the problem reads so downstream tools know how to handle them (or ignore them) appropriately. I will check but I think you can move past these errors.

• AustraliaMember

Hi, when I run picard ValidateSamFile, it gives a different error report:

INFO 2017-06-28 15:26:12 SamFileValidator Validated Read 1,100,000,000 records. Elapsed time: 04:23:53s. Time for last 10,000,000: 106s. Last read position: chrX:143,377,982
INFO 2017-06-28 15:28:46 SamFileValidator Validated Read 1,110,000,000 records. Elapsed time: 04:26:26s. Time for last 10,000,000: 153s. Last read position: chrEBV:121,817
INFO 2017-06-28 15:31:15 SamFileValidator Validated Read 1,120,000,000 records. Elapsed time: 04:28:55s. Time for last 10,000,000: 149s. Last read position: /

## HISTOGRAM java.lang.String

Error Type Count
ERROR:RECORD_OUT_OF_ORDER 2123

What does this error mean? How can I fix it? Thanks for your time!