Friday, October 26, 2012

Sequencing low diversity libraries on Illumina MiSeq

Next Generation sequencing has been used increasingly to investigate sample diversity in amplicon libraries, be it cancer panels, viral species identification or microbial community profiling and has significantly increased estimates of microbial diversity. As the demand has increased to sequence more samples, researchers have looked to the Illumina MiSeq platform for 16S analysis. Using paired-end sequencing at 150 bases (soon to increase to 2 X 250 bases) and generating amplicons of ~300 bp it is possible to overlap data and generate long pseudo-reads. One issue with the Illumina platform is that sequencing libraries of low-diversity can result in low yields and low per-base quality. Nick Loman has developed this simple work-around to spike in a genomic, higher-diversity sample and addresses the three main areas in which low-diversity samples can cause problems on the MiSeq. (1) Focusing, at every cycle. The MiSeq  focuses on the T channel, with a secondary focus to the C channel.  A spike-in of as little as 5% PhiX provides sufficient diversity to prevent any focusing issues irrespective of the library composition. (2) Template building (cycles 1 through 4) and registration (every cycle). Some signal must be present in each channel for RTA to properly do template generation and registration. Again, as little as 5% PhiX prevents these problems as long as cluster density is <700,000. (3) Phasing and matrix estimation, cycles 1 through 12. Average color matrix is measured for cycles 1 through 4, and Phasing is determined for the first 12 cycles. Low diversity samples can cause problems with both if the intensity is not evenly distributed across all channels. A larger spike in of PhiX may be required to address this problem. Details on the configuration used with MiSeq Control Software 1.2.3, and RTA 1.14.23 are given, with the following disclaimer: “This is not a configuration supported by Illumina, so use it at your own risk.”

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