Why "No real operator (M|I|D|N)" in picard?

Question

[dariober]

Hello,

Using picard CollectAlignmentSummaryMetrics I get the error No real operator (M|I|D|N) in CIGAR. I guess this happens when an operator other than M, I, D, N is encountered (and in fact I have soft clipped reads). I can override the error by setting VALIDATION_STRINGENCY=SILENT.

If my guess is correct, I would like to know why CollectAlignmentSummaryMetrics/picard is set to throw an error with operators other than M, I, D, N.

Thanks!

If relevant here's the offending output:

java -jar -Xmx2g ~/applications/picard/picard-tools-1.92/CollectAlignmentSummaryMetrics.jar \
>     IS_BISULFITE_SEQUENCED=True \
>     INPUT=$bam \
>     OUTPUT=${bam%.bam}.AlnSmryMetr.txt \
>     REFERENCE_SEQUENCE=$ref
...
Exception in thread "main" net.sf.samtools.SAMFormatException: SAM validation error: ERROR: Read name M00886:11:000000000-A88VV:1:1109:11516:16954, No real operator (M|I|D|N) in CIGAR
    at net.sf.samtools.SAMUtils.processValidationErrors(SAMUtils.java:448)
    at net.sf.samtools.BAMRecord.getCigar(BAMRecord.java:247)
    at net.sf.samtools.SAMRecord.getAlignmentEnd(SAMRecord.java:456)
    at net.sf.samtools.SAMRecord.computeIndexingBin(SAMRecord.java:1234)
    at net.sf.samtools.SAMRecord.isValid(SAMRecord.java:1644)
    at net.sf.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:540)
    at net.sf.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:522)
    at net.sf.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:481)
    at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:672)
    at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:650)
    at net.sf.picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:109)
    at net.sf.picard.analysis.SinglePassSamProgram.doWork(SinglePassSamProgram.java:55)
    at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:177)
    at net.sf.picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:119)
    at net.sf.picard.analysis.CollectAlignmentSummaryMetrics.main(CollectAlignmentSummaryMetrics.java:92)

And this is the problematic read:

samtools view $bam | grep 'M00886:11:000000000-A88VV:1:1109:11516:16954'
M00886:11:000000000-A88VV:1:1109:11516:16954    83    chr10    3012200    33    49M19S    =    3012200    -49    TAAACAAAATTATAACAAACATCAAACTCTAAATTTAAATAAAAGACCTACAAAAAACATACACTAAA    FGGGFGGGGGGGGFGGGGGGFCGGGGGGGGGGGGGGGGGFGFFGGGGGFGGGGFGGGGGGGGECCCCC    NM:i:0    MD:Z:49    AS:i:49    XS:i:46    RG:Z:grm029_pb_DALIHP.140422.DALIHPplas1_S1_L001_R_001_val_    YC:Z:CT    YD:Z:r
M00886:11:000000000-A88VV:1:1109:11516:16954    163    chr10    3012200    33    68S    =    3012200    49    TAAACAAAATTATAACAAACATCAAACTCTAAATTTAAATAAAAGACCTACAAAAAACATACACTAAA    66ACCGGCFGEGGFGFGFGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFGGGG    AS:i:49    MD:Z:49    NM:i:0    RG:Z:grm029_pb_DALIHP.140422.DALIHPplas1_S1_L001_R_001_val_    XS:i:46    YC:Z:GA    YD:Z:r

Answer 1

[Pierre Lindenbaum]

2nd line: 68S mean that all your read is *ONLY* soft clipped : soft clipped bases of the reads are in 5' or 3' of the read and are not part of the alignment.

An aligned read with a cigar string `68S` makes no sense.

There should have one 'M' or a '=' operator.

[dariober]

Thanks! Of course! I was mislead by the error message. Completely soft clipped reads are the result of clipping overlapping pairs. If overlap is complete one of the two pairs is essentially ignored.