Hello, I have a set of ~ 1 billion gDNA paired reads mapped to a reference genome with BWA-MEM. I tried to use picard's CollectAlignmentSummaryMetrics to generate a summary of the mapping, but it reports zero aligned reads:
FIRST_OF_PAIR 549154663 549154663 1 0 0 0 0 0 0 0 0 0 0 0 99.936337 0 0 0 0 0 0.000001 SECOND_OF_PAIR 549154663 549154663 1 0 0 0 0 0 0 0 0 0 0 0 98.611736 0 0 0 0 0 0 PAIR 1098309326 1098309326 1 0 0 0 0 0 0 0 0 0 0 0 99.274036 0 0 0 0 0 0 A look at the SAM flags manually seems to indicate that most pairs are properly aligned, and the output of samtools flagstat seems to agree: 1102426754 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 duplicates 1097354292 + 0 mapped (99.54%:-nan%) 1102426754 + 0 paired in sequencing 551276790 + 0 read1 551149964 + 0 read2 1042528490 + 0 properly paired (94.57%:-nan%) 1096261806 + 0 with itself and mate mapped 1092486 + 0 singletons (0.10%:-nan%) 36064341 + 0 with mate mapped to a different chr 14493831 + 0 with mate mapped to a different chr (mapQ>=5) I don't mind using samtools flagstat to evaluate the mapping, but I'm puzzled by this and concerned that the problems with the picard output signal further potential issues using picard for duplicate removal, etc. I've seen a few users report similar behavior in the past, and one was able to pin it to their combination of OS and java, but I've tried multiple combinations of java package (Sun, OpenJDK) and picard version and get the same results. To generate the above results I used: Debian 'Wheezy' java version 1.7.0_67 picard version 1.119 samtools version 0.1.18 and the picard command was: java -jar /opt/picard/CollectAlignmentSummaryMetrics.jar MAX_INSERT_SIZE=500 I=bwa.map.w_rg.bam O=bwa.picard.summary As per BWA the library insert size distribution has mean and sd of ~ 180 and 20, respectively. I am attaching a pruned SAM file with just a pair of reads that appear to be mapped correctly but which picard reports as unaligned as per above. Any suggestions or help would be much appreciated. Thanks, Jeremy
test.sam
Description: application/amipro
------------------------------------------------------------------------------ Slashdot TV. Video for Nerds. Stuff that matters. http://tv.slashdot.org/
_______________________________________________ Samtools-help mailing list Samtools-help@lists.sourceforge.net https://lists.sourceforge.net/lists/listinfo/samtools-help
