Try giving it a reference sequence (R=...) to see if the alignment-based
metrics are output.
N
On Thu, Aug 28, 2014 at 2:35 PM, Jeremy Volkening <[email protected]>
wrote:
> 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
>
>
>
>
>
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