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Old 04-28-2016, 01:09 AM   #1
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Location: Denmark

Join Date: Apr 2016
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Default Wierd spikes in library prepared with NEBNext adaptors

I have a problem with some recently prepared libraries (both ChIP and RNA samples). After PCR enrichment spikes occur at around 320bp (within the range of the actual library). I have attached a screenshot of the electropherogram from the FragmentAnalyzer.

Samples were prepared with illumina reagents (TruSeq v2) and after A tailing, samples were size selected to remove upper fragments (0.75x ratio with Ampure beads) and NEBNext adaptors were ligated using NEB reagents and protocol. The libraries were enriched with Q5 mastermix from NEB and adaptors and primers were removed (0.8x ratio with Ampure beads).

Have anyone seen this before? and do you know what it is and whether it will cluster and thus "steal" sequencing.
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Old 04-30-2016, 05:08 PM   #2
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from size distribution plot, your library seems have excess adapters (inefficient ligation), which result in reduced library complexity after PCR, it will "steal" sequencing reads (lots of adapter reads plus spike in reads). Optimizing your ligation step and reducing your PCR cycles can be good troubleshootings.
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