Hi!
I'm considering modifying Illumina TruSeq DNA PCR-Free Library prep kit protocol to produce 1000bp insert size libraries for sequencing on MySeq.
The reason is that the libraries produced will be enriched with custom DNA probes. With larger insert size I plan to get more DNA pulled and sequenced per probe (custom probes are expensive).
We have sequenced Nextera made libraries of such insert size on MySeq without any problems with sequencing before.
I was wondering, if anybody has experience with modifying Illumina TruSeq DNA PCR-Free protocol and would be willing to share his/her experience.
It seems like the logical steps would be to change Covaris settings to produce 1000bp fragments, change the starting concentrations (perhaps from 2ug for 550bp to 3ug), and change the concentrations of SPB in cleanup steps.
Also, since DNA will later be enriched, perhaps fewer cleanup steps would be needed (to preserve sample DNA) as shorter fragments without insert would not be pulled anyhow? Please correct me if I'm wrong.
Thank you,
Lovro
I'm considering modifying Illumina TruSeq DNA PCR-Free Library prep kit protocol to produce 1000bp insert size libraries for sequencing on MySeq.
The reason is that the libraries produced will be enriched with custom DNA probes. With larger insert size I plan to get more DNA pulled and sequenced per probe (custom probes are expensive).
We have sequenced Nextera made libraries of such insert size on MySeq without any problems with sequencing before.
I was wondering, if anybody has experience with modifying Illumina TruSeq DNA PCR-Free protocol and would be willing to share his/her experience.
It seems like the logical steps would be to change Covaris settings to produce 1000bp fragments, change the starting concentrations (perhaps from 2ug for 550bp to 3ug), and change the concentrations of SPB in cleanup steps.
Also, since DNA will later be enriched, perhaps fewer cleanup steps would be needed (to preserve sample DNA) as shorter fragments without insert would not be pulled anyhow? Please correct me if I'm wrong.
Thank you,
Lovro
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