Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, researchers from McGill University sequenced cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix. 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. The researchers 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.

Limit of detection of the ONT MinION platform as calculated from the ERCC experiments 1, 2, 3, 4

A) The number of MinION reads per ERCC transcript from the four ERCC experiments is presented relative to the expected frequency of the corresponding ERCC transcripts from Ambion. A linear regression line is fitted in the data from each experiment. The total number of aligned MinION reads per ERCC experiment along with the regression coefficient values and the correlation coefficient values are presented in the white box legend. The red, green, purple and cyan colored regression lines correspond to the ERCC experiments 4, 2, 1 and 3 respectively. B) Number of total aligned MinION reads obtained from the four ERCC experiments relative to the frequency of ERCC spikes with at least 2 MinION reads. The frequency of ERCC spikes with at least 2 MinION reads was calculated from the regression line equations of picture (A). The number of total aligned MinION reads used are the ones presented in the white box legend of picture