Hi everyone,
We are about to do whole genome sequencing of an 12Mb organism. There are still a lot of "loose ends" in the reference genome, so we aim for long read lengths in order to contribute to the assembly.
Does anyone have experience in 2 x 300bp PE sequncing on the MiSeq, using the TruSeq DNA PCR-Free Library Prep kit (550bp fragments)? Our concern is that the shortest fragments hybridize more easily to the flowchip causing a lot of redundant reads. There will of course be an overlap of 50bp for the 550bp fragments, but will the problem of big overlaps be extensive?
Has anyone used alternative protocols with a more strict size selection or adjusted settings for the fragmentation to get less short reads?
Any help is greatly appreciated!
We are about to do whole genome sequencing of an 12Mb organism. There are still a lot of "loose ends" in the reference genome, so we aim for long read lengths in order to contribute to the assembly.
Does anyone have experience in 2 x 300bp PE sequncing on the MiSeq, using the TruSeq DNA PCR-Free Library Prep kit (550bp fragments)? Our concern is that the shortest fragments hybridize more easily to the flowchip causing a lot of redundant reads. There will of course be an overlap of 50bp for the 550bp fragments, but will the problem of big overlaps be extensive?
Has anyone used alternative protocols with a more strict size selection or adjusted settings for the fragmentation to get less short reads?
Any help is greatly appreciated!
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