Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and...

Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combination with MiSeq platform was applied to target negative- and positive-sense single-stranded RNA viral sequences. This method allowed successful sequence assembly of full or near full length avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) viral genome. Moreover, SISPA analysis applied to unknown clinical cases of mixed viral infections produced genome assemblies comprising 98% NDV and 99% of IBV genomes. Complete or near complete virus genome sequence was obtained with titers at or above 10^4.5 EID₅₀/ml (50% embryo infectious dose), and virus identification could be detected with titers at or above 10³ EID₅₀/ml. Taken together, these studies demonstrate a simple template enrichment protocol for rapid detection and accurate characterization of avian RNA viruses.