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