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

mhmtgenc · 12-17-2018, 04:15 AM

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 · 12-17-2018, 11:39 PM

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 · 12-17-2018, 11:42 PM

Quote:

Originally Posted by 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.

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 · 12-18-2018, 12:03 AM

Possible important issues:

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

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Low diversity on Nextseq 500	MisUser	Illumina/Solexa	9	09-03-2017 03:14 PM
Low number of variants taken from high GB data- NextSeq 500	GizemGoles	Introductions	1	06-21-2017 04:48 AM
low clustering for Sureselect exome v6 on NextSeq	bsvega	Sample Prep / Library Generation	3	06-10-2016 07:57 AM
Questions about whole-exome sequencing on NextSeq 500	newtoseq	Illumina/Solexa	3	11-02-2014 08:26 PM
Experience of Illumina TruSeq Exome coverage?	leesi	Illumina/Solexa	2	03-06-2013 12:45 AM

12-17-2018, 04:15 AM	#1
mhmtgenc Junior Member Location: Turkey Join Date: Nov 2016 Posts: 7	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

12-17-2018, 11:39 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,208	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.

12-18-2018, 12:03 AM	#4
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,208	Possible important issues: 1- degraded or low concentration of blocking oligos 2- inefficient hybridisation step (temperature, time, reaction composition) 2- non-stringent wash steps