Hi
I am working with an NEB kit to prepare Illumina samples. After some troubleshooting with their reps, they recommended an alternate protocol whereby instead of two size selection steps after adapter ligation, I just do two rounds of AMPure bead cleanup.
Bottom line of this is that instead of getting a library from 200-400bp with a beak at ~240bp after the final PCR, I get a smear with roughly the same peak but the width is 200-700bp (see attached figure).
Any thoughts on how this may affect downstream bioinformatics? I need to do two things with the libraries - one is a differential expression analysis between samples; the other is transcriptome assembly and then DE (two completely different experiments)
thanks!
I am working with an NEB kit to prepare Illumina samples. After some troubleshooting with their reps, they recommended an alternate protocol whereby instead of two size selection steps after adapter ligation, I just do two rounds of AMPure bead cleanup.
Bottom line of this is that instead of getting a library from 200-400bp with a beak at ~240bp after the final PCR, I get a smear with roughly the same peak but the width is 200-700bp (see attached figure).
Any thoughts on how this may affect downstream bioinformatics? I need to do two things with the libraries - one is a differential expression analysis between samples; the other is transcriptome assembly and then DE (two completely different experiments)
thanks!
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