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Old 12-17-2018, 04:15 AM   #1
mhmtgenc
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Default Low Exome Coverage with Truseq DNA Exome and NextSeq 500 workflow?

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
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Old 12-17-2018, 11:39 PM   #2
nucacidhunter
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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.

Last edited by nucacidhunter; 12-17-2018 at 11:43 PM.
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Old 12-17-2018, 11:42 PM   #3
mhmtgenc
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Quote:
Originally Posted by nucacidhunter View Post
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.
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Old 12-18-2018, 12:03 AM   #4
nucacidhunter
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Possible important issues:

1- degraded or low concentration of blocking oligos
2- inefficient hybridisation step (temperature, time, reaction composition)
2- non-stringent wash steps
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coverage, nextseq 500, sequencing, targeted coverage, truseq dna exome

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