The Evolution of Human Respiratory Viruses in Peru
Sequencing at the NIAID/JCVI Genome Sequencing Center (GSC) adopts high-throughput pipelines customized to the projects. Viral RNA will be supplied to JCVI by the collaborators. This RNA will serve as template for cDNA generation, amplification and library construction using a variety of Techniques such as SISPA, or long-range PCR with virus specific primers. RTPCR amplicons will be 1.5-2kb and will overlap by at least 200bp. Samples will be sequenced using multiplexed Next Generation (NextGen) sequencing strategies to enable high throughput sequencing of hundreds of barcoded samples. These activities at JCVI include:
- Barcoding cDNA/amplicons from individual viruses;
- Library construction from pools of barcoded DNA;
- High-throughput sequencing with both Ion Torrent and Illumina technologies;
- Deconvolution of barcoded samples and assembly using a software pipeline developed at JCVI;
- Closure of samples using standard Sanger based finishing methods, and finally
- Submission of assembled sequences, meta-data, and sequence files to NCBI.
Additionally, we continue to develop new sequencing approaches with the latest technologies and some of these may be employed to efficiently sequence the viral collection. Robust assembly and automated closure pipelines support validation of assemblies and ensure high quality draft genome sequence generation. The bioinformatics capabilities also allow capture and tracking of various data types, linking genomic information to clinical, geographical, ecological, epidemiological, and associated meta-data as well as sharing and release of the data to collaborators, research groups and public data repositories per the NIAID guidelines.
The following 478 samples, collected in Lima, Piura, and Iquitos, Peru between 2007 and 2012, will be submitted to JCVI for full genome sequencing: 174 human respiratory syncytial virus (RSV). 87 human metapneumovirus (MPV). 217 human parainfluenza type 3 (PIV-3).
This project is proposed as a collaborative study between the NIAID Genome Sequencing Center, the University of Texas Medical Branch, The Connecticut Agricultural Experiment Station, South Florida University, Colorado State University, and the Division of Vector-Borne Diseases, and the Center for Disease Prevention and Control. We will ensure the generation of the best possible data set so further research in the aforementioned areas may be pursued without limitations. The data generated will also be useful for related alphavirus studies.
This project has been funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract numbers N01-AI30071 and/or HHSN272200900007C.