SEQanswers - View Single Post - Amplicon sequencing

eladSeq · 11-28-2012, 12:14 PM

Hi,
We are having trouble sequencing our amplicon pool due to short read failure causing very low pass filter %. For this library, I combined amplicons and then did an agarose gel extraction using a UV gel box. Then followed this with AMPure XP purification.

We have successfully sequenced a similar amplicon pool this way, so I didn't think the UV would cause a problem. The only difference between this library and the previous successful one was pooling more amplicons with different barcodes, so I guess the addition of these new primers could be causing more primer-dimers? On the fragment analyzer we only see a single clean band at 421 bp, but I guess the primer-dimers could be hiding in there by hybridizing to the full length amplicons?

Have you guys had success eliminating the short read failures by reducing the primer-dimers, or eliminating the UV gel extraction step? Any suggestions or advice appreciated. Thanks!

11-28-2012, 12:14 PM	#8
eladSeq Junior Member Location: Baltimore Join Date: Dec 2011 Posts: 9	Hi, We are having trouble sequencing our amplicon pool due to short read failure causing very low pass filter %. For this library, I combined amplicons and then did an agarose gel extraction using a UV gel box. Then followed this with AMPure XP purification. We have successfully sequenced a similar amplicon pool this way, so I didn't think the UV would cause a problem. The only difference between this library and the previous successful one was pooling more amplicons with different barcodes, so I guess the addition of these new primers could be causing more primer-dimers? On the fragment analyzer we only see a single clean band at 421 bp, but I guess the primer-dimers could be hiding in there by hybridizing to the full length amplicons? Have you guys had success eliminating the short read failures by reducing the primer-dimers, or eliminating the UV gel extraction step? Any suggestions or advice appreciated. Thanks!