Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/1/2018
Publication Date: 7/13/2018
Citation: Goraichuk, I., Volkering, J., Afonso, C.L., Suarez, D.L. 2017. Optimization of conditions to sequence long cDNAs from viruses. Meeting Abstract. The American Association of Avian Pathologists and the Avian Veterinary Medical Association Annual Meeting, July 13-17, 2018, Denver, Colorado. 2018 CDROM.
Technical Abstract: Fourth generation sequencing with the Minion nanopore sequencer provides opportunity to obtain deep coverage and long read for single molecules. This will benefit studies on RNA viruses. In the past, Sanger, Illumina, and Ion Torrent sequencing have been utilized to study RNA viruses. Both techniques produce good genome coverage but due to short reads have the inability to determine links among mutations within a genome or to identify quasispecies. Long reads facilitate the identification of associations among mutations and detection mixed infections. Because of the potential for studying other viral and cellular RNAs we optimized sequencing conditions utilizing Avian influenza virus, a multi-fragmented RNA virus with eight different sized genes. cDNAs were PCR amplified with universal primers targeting the highly conserved 5'and 3’ termini of each segment to allow for simultaneous sequencing of all segments. The first strand of cDNA was synthesized using Superscript II, III, and IV Reverse Transcriptase (Invitrogen). All eight genes were amplified by Platinum SuperMix High Fidelity (Invitrogen) for PCR and then subjected to sequencing on a MinION nanopore sequencer. The performance of three cDNA synthesis kits to increase reads coverage and abundance of long-read after sequencing on the MinION was compared to existing methods. Superscript IV RT gave a more uniform distribution of reads across the genome and produced greater coverage of the longest genes. Previous NGS studies NGS produced biased coverage on certain genes (PB2, PB1, and PA genes) at the 5' and 3' termini, with lower coverage in the middle of the gene.