SureSelect target enrichment. Optimal hybridization time

[Staph]

Hello,

We just started using Agilent's sureselect target enrichment system in the lab. Their protocol says hybridization time is 24 hours, however it can be increased to up to 72. Does anyone know if there is a significative difference in the amount captured, or if the eficiency goes down the longer/shorter the hybridization time is?

Thank you!

[ashchin]

Hi ,

I have the same question.
Could you please provide me with any information that you have?

Thanks

[Staph]

hey,

So there is a indeed a correlation between volume after hybridization and capture efficiency. So we got better results after hybridizing for 36 hours instead of 48 as we did at the beginning, and the final volumes of the samples were also higher, about 22-24 ul.
Hope this helps!

[niceday]

we hybridise for 24 hours. We get a good volume back, good coverage and good sequence reads. If you require I might be able to supply percentages.

[ashchin]

Hi Guys,

Thank you for your valuable input.
Could you also please provide the percentages?
Also, what do you use to cover your plate for minimum evporation during 24 hr incubation and during pre- incubations?
I guess yoou use cap during pre incubations.

Thanks

[dnusol]

Hi, sorry to rescue a rather old thread, but I have not found much information on this.
Can anyone comment on the % of reads on target? Has anyone noticed that whole-exome capture gives better % of reads on target than region-capture?

We did a whole-exome capture a while ago and we got about 90% of the reads that passed QC filters aligning in the regions captured, but now with a targeted capture of about 7Mb and only 50% of the QC'ed reads aligned in the targeted regions.

Thanks

Dave

[Heisman]

Hi, sorry to rescue a rather old thread, but I have not found much information on this.
Can anyone comment on the % of reads on target? Has anyone noticed that whole-exome capture gives better % of reads on target than region-capture?

We did a whole-exome capture a while ago and we got about 90% of the reads that passed QC filters aligning in the regions captured, but now with a targeted capture of about 7Mb and only 50% of the QC'ed reads aligned in the targeted regions.

Thanks

Dave

That's somewhat standard, I feel.

[dnusol]

Thanks Heisman,

is there any reasoning behind it? something to do with nonspecific hybridisation?

Cheers

[Heisman]

Thanks Heisman,

is there any reasoning behind it? something to do with nonspecific hybridisation?

Cheers

I don't believe anything has been definitively worked out (although that reason is most likely). One reason is the following: if you were to target the entire genome, your on target efficiency would be 100%. The smaller your target sequence, the "better" your hybridization has to go to work at a certain efficiency percentage.