|Chrzastek, Klaudia - Orise Fellow|
|Lee, Dong-hun - Orise Fellow|
|Sharma, Poonam - Orise Fellow|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/25/2017
Publication Date: N/A
Technical Abstract: Current technologies for next generation sequencing (NGS) have revolutionized metagenomics analysis of clinical samples. One advantage of the NGS platform is the possibility to sequence the genetic material in samples without any prior knowledge of the sequence contained within. Sequence-Independent, Single-Primer Amplification (SISPA) is a primer initiated technique to achieve the non-selective amplification of heterogeneous populations. SISPA is especially useful when the nucleotide sequence of the desired molecule is both unknown and present in limited amounts making its recovery by standard procedures technically difficult. We have modified the SISPA methodology to genome sequencing of RNA viruses. The utility of the method on various types and sources of RNA viruses, obtaining complete genome sequence of avian influenza virus (AIV), Newcastle disease virus (NDV), infections bronchitis virus (IBV) was shown in this study. The detection limit depended on the viral load in the samples. Our results demonstrate complete or near complete virus genome identification with titers at or above 104.5 EID50/ml (50% embryo infectious dose), and virus detection with titers at or above 103 EID50/ml. The application of SISPA - NGS will be discussed, together with its utility for whole genome sequencing of RNA viruses, metagenomics analysis of clinical samples and recovery of low abundance genetic sequences illustrated with AIV, NDV and IBV.