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Strand-specific preparation of full-length PCR-based and PCR-free cDNA libraries by strand-switching

Poster

Date: 2nd May 2017

Strand-specific methods for PCR-based and PCR-free synthesis of full-length cDNA molecules, giving quantitative results, and semi-specific RT-PCR for targeting fusion transcripts 

Fig. 1 PCR-based strand-switching a) laboratory workflow b) ERCC control-mix counts

Strand-switching protocol for preparation of full-length 1D cDNA libraries

We have introduced a PCR-based strand-switching protocol for 1D cDNA sequencing (Fig. 1a). Following first-strand synthesis, reverse transcriptases add 1–3 non-templated Cs to the 3’ end of the cDNA strand. By including an additional primer in the RT reaction which anneals to the non-templated Cs, we can make the reverse transcriptase switch templates, to extend opposite this primer. This incorporates a PCR-priming sequence to the end of full-length cDNAs. After PCR, sequencing adapters are attached. We validated this protocol using the ERCC control mix and obtained excellent correlation with the expected values, regardless of the length of the RNA strands (Spearman’s rho = 0.98; p = 2.6e-61; Fig. 1b), showing quantitative performance.

Fig. 2 PCR-free ‘direct cDNA’ a) laboratory workflow b) ERCC control-mix counts

Direct cDNA protocol for preparation of full-length 1D PCR-free cDNA libraries

It is not necessary to amplify libraries in any way in order to sequence them using our technology. With this in mind, we have developed a PCR-free ‘direct’ cDNA protocol. Here, a first strand cDNA molecule is synthesised, the original RNA is digested, a second strand cDNA is synthesised and sequencing adapters are attached (Fig. 2a). As with the PCR-based protocol, we validated the direct protocol by preparing a library from the ERCC control mix and counting the sequences obtained from each RNA in the mixture. As might be expected from an amplification-free workflow, we obtained a very good correlation with the expected values, regardless of the length of the RNA strands (Spearman’s rho = 0.96; p = 1.4e-52; Fig. 2b).

Fig. 3 Yeast transcriptome a) distance and coverage b) reference coverage c) strand-specificity

Strand-switching showing strand-specificity and enrichment of full-length cDNAs

We applied both PCR and PCR-free protocols to the Saccharomyces cerevisiae S228C transcriptome. Both protocols give good coverage of entire transcripts in single reads (Fig. 2a), and the overwhelming majority of alignments have a coverage value close to 1, indicating that alignments tend to cover full transcripts (Fig. 2b). In both protocols, cDNA extension primes from the eukaryotic poly-A tail, and the signature is retained in the sequences, allowing us to work out the strand in the gDNA from which the transcript was synthesised. This gives us the ability to identify sense and antisense transcripts unambiguously. Fig. 2c shows examples of directionality of full-length reads for a family of transcripts in the SIRV standard panel.

Fig. 4 Targeted detection a) chr22 translocations b) detection protocol c) fusion breakpoint

Sequencing semi-specific RT-PCR products enables characterisation of fusion genes

The q12 region of human chromosome 22 can be involved in several different translocation events (Fig 4a). In each of these, a fusion gene is formed by addition of chromosomal material from one of the fusion partners onto exon 7/8 of the EWSR1 gene on derived chromosome 22. These fusion genes lead to different types of cancer. We reverse-transcribed total RNA from a patient with a known translocation affecting the EWSR1 gene, using a poly TVN primer, and we amplified semi-specifically. The wild-type EWSR1 amplicon and fusion amplicons were 2.2 and 3.1 kb respectively. Sequencing of the amplicons, and alignment to the EWSR1 reference allowed us to pinpoint the position of the breakpoint and to identify the fusion partner. 

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