lortizb
commented
5 years ago Description of my project:
Influenza A virus (IAV) affects a wide range of hosts. Surveillance efforts to understand virus spread and evolution have focused mainly in wild birds and swine due to their role as natural reservoirs and potential source of zoonoses. Even though NGS has had an exponential growth in the last years, platforms like Illumina, still require expensive equipment and costly/time consuming sample preparation. While not as accurate as Illumina sequencing, Oxford Nanopore Technology (ONT) has the potential to increase the speed and quantity of sequence data in remote locations where sequencing core facilities are limited. Using ONT technology in these sites allows to produce real-time sequencing data to rapidly identify the emergence of new variants, including those with the potential to infect poultry and humans.
The goal of this project is to compare the ONT sequencing to our current NGS protocols performed by Illumina. For this purpose, ten viral genomes (8 from wild birds and 2 from swine) previously sequenced by Illumina were sequenced with ONT. These samples were amplified using barcoded Multisegment-RTPCR primers, allowing full genome amplification and barcoding of samples in one step. MS-RTPCR products were pooled for library preparation according to manufacturer´s instructions. Runs were performed using the MinION portable device controlled by MinIT, which contains the pre-configured software with concurrent base calling Guppy and built-in data collection. IAV consensus sequences wil be generated using: Guppy for base calling, Porechop for sample demultiplexing, Canu for viral genome assembly and Nanopolish for polish sequences (in Sapelo2 cluster). Identified subtypes, number of segments and pairwise % identity will be compared with the sequences previously obtained by Illumina.
cbergman
First weekly progress update of my project (description of my project plan)