Agilent SS All Exon V7 kit

[baru]

Anyone here using Agilent SS All Exon V7 kit? I'm continuously getting low yield around 2-3 nM with 100ng input. Any suggestions?

[itstrieu]

Which library and capture kit are you using? I tested the Exon V7 probes with SureSelectXT and SureSelectXT HS a while back. XT gave >50 nM while XT HS gave around 10 nM. Started with 200 ng as the starting concentration.

[baru]

Hey thanks! I am using SS XT Low Input kit with dual index. While I did the test run I got really good yield (around 10-12nM) with 100ng input. I am starting to think it could be reagent issue

[itstrieu]

SS XT Low Input has a similar workflow to HS. After library prep, 1000 ng was inputted into hybridization. Other places where I see can affect yield is lost of the capture beads during the washing step.

Are these the same samples as your test run? They could be DNA of poorer quality.

[baru]

Thanks a lot for your reply. Have you done the SS All Exon V7 dual index library or with MBC? Because the yield with the MBC is ranging from 4-10 nM and for the dual index libraries it goes down to half (1-3nM).Surprisingly, they are consistent throughout the samples. We are working with the Agilent tech support on site and we haven't figured out the explanation yet!!

[itstrieu]

It was with the MBC. You could increase the post-capture PCR cycle by 1 but you might see higher PCR duplicate reads during analysis. I'm curious to see if there are any differences in the sample metrics (% on-target, % duplicates, etc.) and variant calling between the MBC and dual index.

[carladosanjos]

Thanks a lot for your reply. Have you done the SS All Exon V7 dual index library or with MBC? Because the yield with the MBC is ranging from 4-10 nM and for the dual index libraries it goes down to half (1-3nM).Surprisingly, they are consistent throughout the samples. We are working with the Agilent tech support on site and we haven't figured out the explanation yet!!

Did you get your answer from Agilent Tech Support so far? I am experiencing the same issue using the SureSelect HS with dual-index.

Cheers

[baru]

Hello! I'm sorry I missed your query. Well, I did a couple of optimization like increasing the bead capture to 1 hr and reducing the elution volume to 20 ul. Yield slightly improved. So we sequenced them anyway on an SP flow cell before moving with our larger sets of samples. Exome quality was extremely good in the end. So don't worry about the yield, as long your libraries are of good quality go ahead and sequence them
ps: This is just my experience