I'm making Nextera libraries on mosquito genomic DNA using Illumina's Nextera kit. I ran the protocol exactly as stated and ran my resulting libraries on a Bioanalyzer HS DNA chip. My insert sizes are larger than I expected (~1000bp observed versus 300-500bp expected) [picture attached--apologies for the crude display of fragment size].
I spoke with Illumina's technical support and they suggested that this was due to:
- Too much starting material
- Tagmentation incubation too short.
- Tagmentation incubation not warm enough.
The Illumina protocol states that "libraries with an average size >1kb may require clustering at several concentrations to achieve optimal density," which suggests to me that this isn't a problem and that the libraries can still be sequenced with a bit of optimization.
We're aligning back to a reference sequence so I don't think large insert sizes will be a problem from the bioinformatic perspective (in fact, they may help with mapping!).
Is this something I should be concerned with?
I spoke with Illumina's technical support and they suggested that this was due to:
- Too much starting material
- Tagmentation incubation too short.
- Tagmentation incubation not warm enough.
The Illumina protocol states that "libraries with an average size >1kb may require clustering at several concentrations to achieve optimal density," which suggests to me that this isn't a problem and that the libraries can still be sequenced with a bit of optimization.
We're aligning back to a reference sequence so I don't think large insert sizes will be a problem from the bioinformatic perspective (in fact, they may help with mapping!).
Is this something I should be concerned with?
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