Low Exome Coverage with Truseq DNA Exome and NextSeq 500 workflow?

[mhmtgenc]

Dear All,

I studied a whole exome with Truseq DNA Exome (2x75 and suitable High output kit) on NextSeq 500.. AFter sequencing I got around 46 million reads per sample and 42 million of these were mapped as globally.

As you guess I did BamQC report and got that only 11 Milllion of these 46 million reads are mapped to the targets of TruSeq DNA Exome kit. And I have used the bed file provided by Illumina for this kit.

So what could be the possible problem that On global Nearly 90% of my reads are mapped but in target only around 23% of the reads are mapped?

Thanks in advance

[nucacidhunter]

It indicates that on target reads (enrichment) is low and most of reads are from background. This figure for most kits is above 70%. At least one step in target capture has been non-optimal.

I wonder what is the duplication rate.

[mhmtgenc]

It indicates that on target reads (enrichment) is low and most of reads are from background. This figure for most kits is above 70%. At least one step in target capture has been non-optimal.

Thanks for the answer. So could you suggest anything with that? Because I have been following the exact steps of the kits checklist guide.

[nucacidhunter]

Possible important issues:

1- degraded or low concentration of blocking oligos
2- inefficient hybridisation step (temperature, time, reaction composition)
2- non-stringent wash steps