From QC to isoform characterization : Evaluation and improvements of Nanopore sequencing in a RNASeq context

Sophie Lemoine (IBENS)
Thursday, June 6, 2019 - 10:30 to 11:00
Room Aurigny
Talk abstract: 

Transcript identification is a real challenge with short read sequencing. With Oxford Nanopore Technologies (ONT), our aim is to sequence full-length cDNA to directly access isoforms. We have successfully validated analysis of differential expressed genes on a mouse model of myelination blockage following the standard ONT protocol. The mean length of our reads was 1.2kb, which is lower than the estimated 2kb mean length of the transcripts and even worse if we consider the TSL1 tagged transcripts (2.6kb). To improve our results, we combined SmartSeq and ONT technologies to synthesize full-length cDNA from total RNA. The cDNA were barcoded in order to sequence multiplesamples on a single MinION run and allow differential expression analyses. The SmartSeq/ONT protocol allowed us to sequence much longer cDNAs. The mean length of thereads was then about 2.6kb and the small reads that were the majority of the population with ONT protocol were eradicated. We were able to detect more differentially expressed targets. The targets detected were longer than the ONT protocol ones. The optimized protocol globally achieved a better 5’-3’ transcripts coverage and not surprisingly, for those longer than 2kb. If it does not ensure you have full-length cDNAs, it can be reliable for cDNA sequencing and improve isoform annotation andquantification using dedicated pipelines, such as FLAIR or Pinfish.The goal of my talk is to give an idea of :- the evolution of the protocols tested and improved;- the developments we had to perform to make the QC of our runs;- the ongoing evaluation of FLAIR and Pinfish in our context.