Here, 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. Similar to PacBio RS II, the ONT MinION has been shown that can resolve the exon structure of mRNA molecules transcribed from genes with a large variety of isoforms 10. Recently, another long-read DNA sequencing technology based on nanopore sequencing (MinION) was introduced from Oxford Nanopore Technologies Ltd (ONT) 9. The PacBio RS II 7 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. Uneven read coverage 4, complex splicing 5 and potential sequencing bias 6 complicates even more the task. Nevertheless, inferring alternatively spliced isoforms of genes from short read data through statistical assignment of the most probable combination of exons is still computationally challenging and not very accurate 3. Transcriptome sequencing using short read technologies (Illumina HiSeq 2500 1, Ion Proton 2) provides valuable information on transcript abundance, rare transcripts and variable transcription start or end sites. This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (r pearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (r pearson = 0.75). 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. The majority of cDNAs were sequenced as full length. No length or GC content bias was observed. ![]() The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. 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.
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