Ken Heuermann, Brian Ward, Dom Fenoglio
Since the publication of this article, we have launched a WTA2 NGS compatible kit, SEQR, that includes primer removal.
Whole Transcriptome Amplification (WTA2) 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, 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 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 amplification of RNA isolated from samples of severely restricted quantity or quality.
Strategies for the integration of 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.
Complete WTA2 kit has been demonstrated to effectively amplify damaged and/or low quantities of RNA template1-13. Exponential PCR-based amplification provides both sensitivity and reproducibility, while maintaining relative levels of transcript abundance1,2,4,8,10-12.
The most common misconception, detracting from the general acceptance of PCR-based amplification strategies, is the comparison of the disjointed increase in relative levels of two different transcripts during amplification in the same reaction—due primarily to differences in amplicon length and complexity. This is not a legitimate argument. WTA2 amplification technology addresses this misconception through the following points:
The non-self complementary primer sequence is the linchpin of amplification, critical for efficient, positionally unbiased amplification of each transcript and comprehensive coverage of the transcriptome. The 5’ universal sequence spans 22 nucleotides, with the quasi-random 3’ region comprising 10 nucleotides (WTA2 Workflow). Because of the significant cost of labeled nucleotides and characteristic short read lengths for the Illumina and ABI platforms (Table 1), it is cost-effective to remove these primer sequences prior to NGS sequencing.
Several enzymatic processes for primer sequence removal are under consideration.
Click on image below for larger view.
An alternative to enzymatic manipulation of the WTA2-amplified library is the following.
The expectations of researchers are driving further development of methodologies and technology for single-cell analyses. Examples of RNA-Seq24-26 and RIP-Seq27 analyses (RNA Immunoprecipitation) are becoming more prevalent, in the study of disease and development, particularly in the assessment of oocyte and embryo development28.
WTA2 produces 3 to 5 µg of amplified product from 20 picograms of input total RNA (a singlecell equivalent quantity).
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