SAM validation errors

[ashwatha]

Hi,

I need some help understanding these errors when I use picard to validate a SAM/BAM file:


$java -jar picard-tools-1.50/ValidateSamFile.jar I=somebam.bam
ERROR: Read groups is empty
ERROR: Record 38, Read name V15-13:3:73:1694:1074, CIGAR should have zero elements for unmapped read.
ERROR: Record 37, Read name V15-13:3:73:1694:1074, Mate negative strand flag does not match read negative strand flag of mate

What does this error mean? I understand why an unmapped read should have zero elements in the CIGAR string, but reads at records 37 and 38 seem to be mapped. Can someone help me understand this error? These are the corresponding records from the SAM file:

V15-13:3:73:1694:1074 89 chr1 325709 0 69S46M = 325709 0 GTGGCTGACTATGGAAGCCCAAGTCTTCCTCAAATCCGGCTTTTGCCCAACTTCTGACTACTGTCGGACTCTACAGGTCAGCCTCTGCCTCACAGTGGACCCTCCAGACCCAGAT ######################################################################CA,ECEA<EC>AA+BAABB+EC4EGGGGDD<D8BFGDDGGGGGEG XC:i:46 XT:A:R NM:i:0 SM:i:0 AM:i:0 X0:i:5 X1:i:13 XM:i:0 XO:i:0 XG:i:0 MD:Z:46

V15-13:3:73:1694:1074 181 chr1 325709 0 58M57S = 325709 0 TTCTGGCAGGAGGACTTTTGGCGCCGACATTGGCCGCCAGGCGCTAGACAGGCCCTGGGCCAAAAAAGGCTGTTGTTAGGCAGCGGTTGTGCCGGTGGCCCCAGCCAAGAGGAAG ##########################################################??37B<?35991*9773,+>8@=7>228?B,B775''=-B,BDDGG?EB:F=?@BEE XC:i:58

Thanks!
-- Ashwatha.

[av_d]

READ: V15-13:3:73:1694:1074
FLAG: 89
(read paired, mate unmapped, read reverse strand, first in pair)

http://picard.sourceforge.net/explain-flags.html

RNAME: chr1

MRNM: = (? that should not be)

READ: V15-13:3:73:1694:1074 (mate)
FLAG: 181
(read paired, read unmapped, read reverse strand, mate reverse strand, second in pair)

RNAME: chr1 (? but this read in not mapped)


you can fix them using FixMateInformation of Picard, otherwise use VALIDATION_STRINGENCY=SILENT

[ashwatha]

Thank you for the help.