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CollectHsMetrics for merged bams
I am using PICARD version 2.1.1 to generate metrics for my samples.
The samples were sequenced with llumina NextSeq, we loaded a single library that is a pool of 12 samples.
The flow cell has 4 lanes so at the end of de-multiplexing we have 4 paired fastq files per sample.
I aligned them separately with BWA and then I merged the 4 bams with MarkDuplicates in one step as suggested in your guidelines.
Read groups assigned:
@RG ID:13-2250_L001 LB:L001 PL:illumina SM:13-2250_L001 PU:170129
@RG ID:13-2250_L002 LB:L002 PL:illumina SM:13-2250_L002 PU:170129
@RG ID:13-2250_L003 LB:L003 PL:illumina SM:13-2250_L003 PU:170129
@RG ID:13-2250_L004 LB:L004 PL:illumina SM:13-2250_L004 PU:170129
After the CollectHsMetrics I obtained a MEAN_TARGET_COVERAGE of 165 per sample that is far away from the results expected. We used a high output flow cell so we expected to double (at least) that value.
And in fact, looking at the illumina report generated by base space it is indicated a mean coverage of 400x.
Am I doing something wrong? Is there some step that I am missing?
Any help is really appreciated.