| Autor: |
Keller MW; Oak Ridge Institute of Science and Education (ORISE), Oak Ridge, Tennessee, USA., Rambo-Martin BL; Battelle Memorial Institute, Atlanta, Georgia, USA., Wilson MM; Battelle Memorial Institute, Atlanta, Georgia, USA., Ridenour CA; Battelle Memorial Institute, Atlanta, Georgia, USA., Shepard SS; Influenza Division, National Center for Immunization and Respiratory Diseases (NCIRD), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA., Stark TJ; Influenza Division, National Center for Immunization and Respiratory Diseases (NCIRD), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA., Neuhaus EB; Influenza Division, National Center for Immunization and Respiratory Diseases (NCIRD), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA., Dugan VG; Influenza Division, National Center for Immunization and Respiratory Diseases (NCIRD), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA., Wentworth DE; Influenza Division, National Center for Immunization and Respiratory Diseases (NCIRD), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA., Barnes JR; Influenza Division, National Center for Immunization and Respiratory Diseases (NCIRD), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. fzq9@cdc.gov. |
| Abstrakt: |
For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID 50 6.8 × 10 9 ), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods. |
