Abstract
To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.
Highlights
Transcriptome sequencing using short read technologies (Illumina HiSeq 25001, Ion Proton2) provides valuable information on transcript abundance, rare transcripts and variable transcription start or end sites
The different types of reads produced from the Oxford Nanopore Technologies Ltd (ONT) MinION platform has been described in detail in other studies[11]
To assess whether in the case of the HEK-293 cDNA library run on the ONT MinION platform, a flow cell specific behavior affected the full length sequencing of at least the template strand of the cDNA molecules, we introduced into the HEK-293 total RNA, three selected RNA transcripts (“Spike-1”, “Spike-4”, “Spike-7”) at different concentrations
Summary
Transcriptome sequencing using short read technologies (Illumina HiSeq 25001, Ion Proton2) provides valuable information on transcript abundance, rare transcripts and variable transcription start or end sites. The PacBio RS II7 zero–mode waveguide (ZMW) long-read sequencing technology has proven capable of characterizing the transcriptome in its native, full-length form unravelling novel gene isoforms not previously observed in RNA-seq experiments[8]. Another long-read DNA sequencing technology based on nanopore sequencing (MinION) was introduced from Oxford Nanopore Technologies Ltd (ONT)[9]. We assessed the ONT MinION performance for its ability to sequence the cDNAs with good accuracy in terms of cDNA abundance, sequence identity and in full length. Can sequence quantitatively cDNA molecules similar with the Illumina and PacBio RS II platforms paving the way for full length sequencing of cDNA molecules with nanopores
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