Thursday, August 19, 2010

Using Nextera technology for RNA-Seq expression analysis


We’ve had several inquiries about using Nextera™ technology with cDNA, in order to generate libraries for RNA-Seq and subsequent gene expression analysis. We are grateful to the Richard M. Myers Laboratory at the Hudson Alpha Institute for Biotechnology (Huntsville, AL), for testing this application of Nextera technology. They used the following strategy (click on all figures to enlarge them):

Strategy for library preparation

With this strategy, decreased coverage at the ends of cDNAs is expected because two transposome insertions are required to produce a functional sequencing template.

Libraries using human RNA (ECC-1) were prepared with both the Nextera workflow and a standard RNA-Seq workflow, and sequenced on the Illumina GAII platform. Two Nextera libraries were prepared: one from 50 ng of cDNA, and the other from only 10 ng of cDNA. The results from both the conventional RNA-Seq and Nextera procedures compare very well:

Relative gene expression analysis


The data show that Nextera technology yields representative libraries, even with very low amounts of cDNA:

Correlation based on sample input amount


As seen from the coverage of the cDNA termini and library complexity, both libraries were highly complex and representative of the transcripts. The red arrows show the expected drop in coverage of 5' and 3’ ends with the Nextera library:

Library coverage and complexity

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