+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

The effect of preprocessing by sequence-independent, single-primer amplification (SISPA) on metagenomic detection of viruses

The effect of preprocessing by sequence-independent, single-primer amplification (SISPA) on metagenomic detection of viruses

Biosecurity and Bioterrorism 11(Suppl. 1): S227

Compared to routine diagnostics, screening for pathogens in outbreak situations, with or without intentional release, poses demands on the detection technology to not only indicate the presence of already known causative agents but also novel and unexpected pathogens. The metagenomic approach to detecting viral pathogens, using unbiased high-throughput sequencing (HTS), is a well-established methodology with a broad detection range and wide applicability on different sample matrices. To prepare a sample for HTS, the common presequencing steps include homogenization, enrichment, separation (eg, magnetic separation), and amplification. In this initial study, we explored the benefits and drawbacks of preprocessing by sequence-independent, single-primer amplification (SISPA) of nucleic acids by applying the methodology to artificial samples. More specifically, a synthetic metagenome was divided into 2 samples, 1 unamplified and 1 diluted, and amplified by SISPA. Subsequently, both samples were sequenced using the Ion Torrent Personal Genome Machine (PGM), and the resulting datasets were analyzed by using bioinformatics, short read mapping, de novo assembly, BLAST-based taxonomic classification, and visualization. The results indicate that even though SISPA introduces a strong amplification bias, which makes it unsuitable for whole-genome sequencing, it is still useful for detecting and identifying viruses.

Please choose payment method:

(PDF emailed within 0-6 h: $19.90)

Accession: 056309272

Download citation: RISBibTeXText

PMID: 23971810

DOI: 10.1089/bsp.2013.0008

Related references

Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses. Virology 509: 159-166, 2017

Single primer isothermal amplification (SPIA) combined with next generation sequencing provides complete bovine coronavirus genome coverage and higher sequence depth compared to sequence-independent single primer amplification (SISPA). Plos one 12(11): E0187780, 2017

Sequence independent single primer amplification sispa utility and application to the detection of enterically transmitted non a non b hepatitis et nanbh. Journal of Cellular Biochemistry Suppl. (13 Part E): 293, 1989

Sequence-independent, single-primer amplification (SISPA) of complex DNA populations. Molecular and Cellular Probes 5(6): 473-481, 1991

Sequence-specific, single-primer amplification and detection of PCR products for identification of hepatitis viruses. Journal of Virological Methods 61(1-2): 127-134, 1996

Detection of TT virus by single-primer sequence-independent amplification in multiple samples collected from an outbreak of gastroenteritis. Archives of Virology 154(6): 981-985, 2009

Optimisation of a single-primer sequence-independent amplification (SP-SIA) assay: detection of previously undetectable norovirus strains associated with outbreaks of gastroenteritis. Journal of Virological Methods 158(1-2): 30-34, 2009

Circular genomes related to anelloviruses identified in human and animal samples by using a combined rolling-circle amplification/sequence-independent single primer amplification approach. Journal of General Virology 88(Pt 10): 2696-2701, 2007

Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture-Based Isolates. American Journal of Tropical Medicine and Hygiene 96(2): 389-394, 2017

Oligonucleotide-based microarray for detection of plant viruses employing sequence-independent amplification of targets. Journal of Virological Methods 163(1): 57-67, 2010

Amplification for Whole Genome Sequencing of Bacteriophages from Single Isolated Plaques Using SISPA. Methods in Molecular Biology 1681: 165-178, 2018

Development and evaluation of a novel nucleic acid sequence-based amplification method using one specific primer and one degenerate primer for simultaneous detection of Salmonella Enteritidis and Salmonella Typhimurium. Analytica Chimica Acta 770: 169-174, 2013

Profile hidden Markov models for the detection of viruses within metagenomic sequence data. Plos one 9(8): E105067, 2014

Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses. Applied and Environmental Microbiology 77(21): 7663-7668, 2011

Detection of Mobiluncus species by special primer amplification and 16S DNA sequence analysis. Wei Sheng Wu Xue Bao 45(1): 27-30, 2005