The Picard FilterSAM READ is throwing error

Question

[a.james]

Dear All,

I am using Picard FilterSamReads for filtering a list of sorted reads from a read_name/_id sorted BAM file. I am wanna sort the list of reads which are of certain mapping quality.

Here is what I tried,

java -jar -Xmx4G /path/picard-2.18.4-0/picard.jar FilterSamReads I=simulate_50X_pat_sorted_read.bam O=simulate_50X_pat_sorted_quality_pat.bam READ_LIST_FILE=pat_quality2 FILTER=includeReadList

And the above line is throwing the following error,

INFO    2019-11-19 16:51:40 FilterSamReads  Filtering [presorted=true] simulate_50X_pat_sorted_read.bam -> OUTPUT=simulate_50X_pat_sorted_quality_pat.bam [sortorder=queryname]
ERROR   2019-11-19 16:51:40 FilterSamReads  Failed to filter simulate_50X_pat_sorted_read.bam
java.lang.IllegalArgumentException: Alignments added out of order in SAMFileWriterImpl.addAlignment for file:///cluster/work/projects/simulate_50X_pat_sorted_quality_pat.bam. Sort order is queryname. Offending records are at [1-93166-:1-93466--:::::::::::52438MzI4MTAy:1] and [1-102419--:1-102119-:::::::::::199592NzM1MA==:1]
    at htsjdk.samtools.SAMFileWriterImpl.assertPresorted(SAMFileWriterImpl.java:213)
    at htsjdk.samtools.SAMFileWriterImpl.addAlignment(SAMFileWriterImpl.java:200)
    at picard.sam.FilterSamReads.filterReads(FilterSamReads.java:256)
    at picard.sam.FilterSamReads.doWork(FilterSamReads.java:345)
    at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:282)
    at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103)
    at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113)
[Tue Nov 19 16:51:40 CET 2019] picard.sam.FilterSamReads done. Elapsed time: 0.20 minutes.
Runtime.totalMemory()=3706716160

I have tried to remove the above reads from my input list and tried again, however, the same ERROR message appeared with different read names.

I would like to know, what else other than sorting the inputs should be done before running the FilterSamReads tool?

Any help or suggestion is much appreciated.

Answer 1

[Pierre Lindenbaum]

was the sam sorted with samtools ? it might be that error: https://github.com/samtools/hts-specs/issues/5

try

samtools view -h simulate_50X_pat_sorted_read.bam |\
grep -v '^@HD'  |\
java -jar -Xmx4G /path/picard-2.18.4-0/picard.jar FilterSamReads I=/dev/stdin O=simulate_50X_pat_sorted_quality_pat.bam READ_LIST_FILE=pat_quality2 FILTER=includeReadList

[a.james]

Thanks for the reply. It was previously sorted on the read_id using samtools sort -n.

[Pierre Lindenbaum]

yeah, the order between samtools and picard is not the same, that's why picard crashed. https://github.com/samtools/hts-specs/issues/5#issuecomment-29213725