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Hello everyone! I am performing library generation of samples to be sequenced on Illumina HiSeq. I use kapa lib prep protocol and NimbleGen SeqCap EZ protocol to capture my target. Unfortunately, I end up having poor amplification after postCap PCR (1-2 ng/ul) and I am confused about what causes this poor library yield. Any suggestion?
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There are a lot of things that can cause poor yield after hybridization. What was your pre-cap LM-PCR yield? How much sample did you put into the hybe?
Are you using the Roche Hybe and Wash kit with both Hybe buffer and Component A? Are you using a heated lid on the PCR machine during the hybe? Did you make sure the lid was set to 10 degree offset? What was the volume of the reaction when you took down the hybe? It should be close to 15 ul.
If you got sufficient DNA and put the correct amount of sample into the hybe, you should look at the hybe step. Assuming you are hybridizing at the correct temperature, you should make sure the streptavidin beads are never completely drying out during the washing steps. It's also important that the beads be washed with all the buffers at the correct stringency.
Did you by any chance include a PCR control in the post-cap LM-PCR step?
Roche Tech Support is pretty good with helping out, please give them a call if you keep having problems.
For immediate, contact the Sequencing Solutions Hotline:
00800-73783623 or +49 621 7650 77721 -
Is this for human? We've seen similar post-cap post-pcr DNA amounts when doing capture in a large-genome organism (30gb).Comment
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The precap yield was 1.8 micrograms and i used 1 microgram for hybridization as recommended. The reaction is performed at 47 degrees with lid at 57 degrees. The volume of the reaction was 14.5 after the incubation and the wash steps were performed following the protocol very carefully. I really do not understand where the problem is...Comment
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What is the size of the capture?
Did you include the streptavidin beads in the PCR?Comment
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The size of the target region is 194kb.
I do perform with-bead-protocolComment
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How do you clean up after PCR?Comment
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Hi thiNGS,
194kb is a small-ish target, and that alone can reduce the yield a bit (depleting the input library of capturable library fragments limits the post-capture PCR's template). How many post capture PCR cycles are you running, and what method are you using to quantitate?
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