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Low cluster density of Truseq "high throughput" libraries - both DNA and RNA
Hi genomics folks,
Has anyone got any experience in clustering Illumina Truseq RNA 'HT' (high throughput) and Truseq DNA HT libraries?
I work in an NGS service, and we recently tried out the new Truseq HT kits. I like the idea of multiplexing up to 96 Truseq libraries within a single lane.
These libraries were quantified by qPCR following our normal protocol for Truseq libraries, using the KAPA library quant kit. They were then clustered using a cbot -> HiSeq or straight on to the MiSeq. The resulting cluster density was 1/4 to 1/2 of what we would expect, on both machines.
These projects were unrelated and the library prep was performed by very experienced guys in the lab - I'm starting to wonder if the HT kits require different clustering conditions. According to Illumina, these libraries should cluster OK using the normal protocol.
Has anyone else experienced this problem?
Has anyone got any experience in clustering Illumina Truseq RNA 'HT' (high throughput) and Truseq DNA HT libraries?
I work in an NGS service, and we recently tried out the new Truseq HT kits. I like the idea of multiplexing up to 96 Truseq libraries within a single lane.
These libraries were quantified by qPCR following our normal protocol for Truseq libraries, using the KAPA library quant kit. They were then clustered using a cbot -> HiSeq or straight on to the MiSeq. The resulting cluster density was 1/4 to 1/2 of what we would expect, on both machines.
These projects were unrelated and the library prep was performed by very experienced guys in the lab - I'm starting to wonder if the HT kits require different clustering conditions. According to Illumina, these libraries should cluster OK using the normal protocol.
Has anyone else experienced this problem?