Modification of the Transplex WTA2 Amplification Product for Next Generation Sequencing

摘要

Transplex Whole Transcriptome Amplification (WTA2)a exponentially amplifies RNA producing a double-stranded cDNA library while precisely maintaining differential levels of individual transcripts in test and reference samples. Though originally designed to amplify nanogram quantities of RNA, Transplex WTA2 has been shown to be exceedingly effective for amplification from damaged RNA template (FFPE and laser captured tissue samples) and single-cell input quantities (picograms). The efficacy of Transplex WTA2 amplification for downstream applications, primarily qPCR and expression microarray analysis, is well-documented. It follows that the utilization of next-generation sequencing for gene expression research and diagnostics would be well served by Transplex amplification of RNA isolated from samples of severely restricted quantity or quality. Strategies for the integration of Transplex WTA2 with next-generation sequencing are examined, with particular emphasis on elimination of the characteristic fixed primer sequence associated with each amplicon in the amplification library. Removal of these sites will allow direct entry of the resulting product into the sequencing workflow. Methods under consideration will enable the WTA2 amplicon to feed into the current sample prep protocols for the Illumina GA and GAII, SoLiD 5500/5500xl, and Roche-454 GS FLX/Junior platforms.