A recent run of a very short insert library (average insert size 32bp) on a lane of Hiseq 3000 yielded only 50 million reads. Many clusters were excluded by the chastity filter due to mixed populations. We suspect the kinetic exclusion amplification step in the patterned flow cells used in Hiseq 3000 and 4000 to work less well for such short inserts, hence more clusters are mixed and excluded. We wondered if lowering the input molarity could improve the yield for such libraries and if anybody had experience with this and could give advice as to what molarity works best. Any help is much appreciated.
Many thanks,
moe
From Illumina's patent: "Accordingly, kinetic exclusion can be achieved in such embodiments by using a relatively slow rate of transport. For example, a sufficiently low concentration of target nucleic acids can be selected to achieve a desired average transport rate, lower concentrations resulting in slower average rates of transport."
(aDNA, ancient DNA, HTS, high-throughput sequencing, NGS, next-generation sequencing)
Usually, submits libraries with concentrations of 5 nM or higher is optimal. But in such cases prepare of the library has the direct effect on the result. I think you should focus to prepare the library.
dear Moritz Muschick
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