- Paynter, Ken; Afonso, Claudio
- USDA - Agricultural Research Service
- SeqWright DNA Technology Services
- Start date
- End date
- SeqWright and Southeast Poultry Research Laboratory (SEPRL) wish to collaborate on a project to sequence 1000 complete genomes of Avian Influenza. SeqWright will research and develop reagents (primers) for rapid sequencing of avian influenza and will create methods for rapid annotation of the genome. SEPRL will utilize the annotated genomic information corresponding to 1000 avian influenza isolates to study epidemiology and evolution.
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- APPROACH: SEPRL will isolate viral RNA from our collection of 1000 avian influenza viruses and the viral RNA will be sent to Seqwright. Seqwright will assign a scientist to analyze previously known avian influenza genomic sequences and to design (using primer design software) sufficient sets of primers for amplification and sequencing of all genes from all different serotypes existing in our collection. Primers will be used to generate cDNAs, and for bi-directional DNA sequencing (performed by automated ABI Prism? 3730xl DNA sequencers). Seqwright will score \mutations/heterozygotes/ heteroindels and will assign an expert curator to review and re-sequence (if necessary) all called mutations to confirm their accuracy. Results containing the raw data, full-length consensus text sequence, and genotype calls will be provided to SEPRL. SEPRL will review the data and analyze evolutionary and phylogenetic relationship among isolates.
PROGRESS: 2006/09 TO 2008/09
With the purposes of obtaining full coding genome sequences of the Southeast Poultry Research Laboratory (SEPRL) collection of avian influenza viruses we have conducted ribonucleic acid (RNA) isolation from 1000 isolates and initiated the following steps on all of the samples: 1) One step real-time reverse transcriptase-polymerase chain reaction (RT- PCR), 2) Quality control (QC) of RT-PCR Products, 3) Sequencing, 4) Alignment and editing of sequence data, 5) Data alignment, and 6) Sequence verification and quality control. Steps 2 to 5 have been performed at SeqWright with steps 1 and 6 at SEPRL. Due to sample variability within waterfowl isolates, multiple rounds of RT-PCR and sequencing have been performed for some isolates to obtain complete double stranded sequence coverage whenever possible. In addition all sequences are evaluated for quality at SEPRL and completely verified sequences are submitted to Genbank. A total of 4974 genes have been completely sequenced and 1203 have been partially sequenced out of a total maximal number of 8000 genes. The distribution of the genes that have been completely sequenced per batch is as follows: Batch numbers 1, 2, 3, 4, 5, 6, and 7 had a total of 91, 84, 607, 1189, 9, 555 and 2264 genes completely sequenced and verified art SEPRL respectively, producing a final grand total of 4,974 genes. Seqwright has been having difficulties sequencing the hemagglutinin (HA) and neuraminidase (NA) genes due to the greater genetic variability and the lack of amplification during polymerase chain reaction (PCR), while less problems are found in the characterization of Polymerase Basic 2 (PB2), Polymerase Basic 1 (PB1), Polymerase A, nucleoprotein (NP), Matrix 1, and Non- structured genes. Seqwright has informed United States Department of Agriculture (USDA) that the cost of sequencing genomes has increased above their expectations, mainly due to the lack of amplification in some isolates.
MONITORING: One primary person at SEPRL and SeqWright work together to conduct weekly trouble shooting for difficult or mistyped isolates, ensuring full double stranded coverage, and manual checking of the data for ambiguities, gaps, and frameshifts. Tracking of data is performed at SEPRL and SeqWright, as well as compilation of the data into a useable format for internal use at SEPRL. Translation is confirmed when the sequence is complete. Conference calls and internal meetings are also conducted to report on progress and when any changes are needed in the protocols.
- Funding Source
- Agricultural Research Service
- Project number
- Accession number
- Prevention and Control
- Chemical Contaminants
- Preventive Food Safety Systems
- Meat, Poultry, Game