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Old 10-23-2019, 04:46 AM   #1
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Location: Michigan

Join Date: Aug 2012
Posts: 6
Default MiSeq tolerated library size distribution

Our lab is trying some new library generation protocols where we are size selecting a much broader range of libraries than we have run previously, a 200 bp span as opposed to a 20 bp span. When we tried sequencing this pool on MiSeq, v2 300 cycle kit single-end, the run gave an out of focus error about halfway through. It seems like this corresponds with something seen in our bioanalyzer traces, that the libraries are not evenly distributed across the size range but heavier on the smaller side. My thought was that once all these smaller libraries were sequenced, there wasn't enough larger libraries left for the MiSeq to maintain good focus? PhiX was included at 5%.

Is there a maximum tolerated spread of library sizes in a MiSeq sequencing run? Would simply spiking in a higher percent PhiX resolve the issue (10 or 25%)?

We are planning to change to paired-end sequencing moving forward as another method to hopefully help with this issue.
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