We have recently switched to sequencing paired end 250, and noticed that there is always a bias towards the smaller fragments. We made sure that our libraries are in the exact size range that we wanted (with a lot of size selection experiments), and all of the QC shows that we achieved this range. The problem is that the reads we are getting are all from the lower range so our larger fragments aren't being as well represented. Any thoughts or similar problems?
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Sure, that is what we see also. The only way to reliably see larger amplicons is to get rid of all the shorter amplicons.
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Phillip -
What do you use to completely remove smaller amplicons? We currently do bead size selection and seem to be removing the majority of the smaller fragments, but they still make up the majority of our reads.Comment
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Ampure. We use just use a lower amount of Ampure, or multiple extractions.
I think the trick is to look at your results on the bioanalyzer trace but pay attention only to the left side of the curve. Where it reaches the baseline and maybe 100 bases larger will be where about 50% of your reads will be. (Just guessing on the percentage, but that is the idea.)
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PhillipLast edited by pmiguel; 07-01-2015, 06:58 AM.Comment
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