Sequencing viral genomes from a single isolated plaque

Jessica Depew,David E Wentworth,Bin Zhou,Derrick E Fouts,Galina Koroleva,Jamison M Mccorrison,Janaki Purushe

doi:10.1186/1743-422x-10-181

Jessica Depew, David E Wentworth + Show 5 more

Open Access

https://doi.org/10.1186/1743-422x-10-181

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Journal: Virology journal	Publication Date: Jun 6, 2013
Citations: 39	License type: cc-by

Affiliation: J. Craig Venter Institute

Abstract

BackgroundWhole genome sequencing of viruses and bacteriophages is often hindered because of the need for large quantities of genomic material. A method is described that combines single plaque sequencing with an optimization of Sequence Independent Single Primer Amplification (SISPA). This method can be used for de novo whole genome next-generation sequencing of any cultivable virus without the need for large-scale production of viral stocks or viral purification using centrifugal techniques.MethodsA single viral plaque of a variant of the 2009 pandemic H1N1 human Influenza A virus was isolated and amplified using the optimized SISPA protocol. The sensitivity of the SISPA protocol presented here was tested with bacteriophage F_HA0480sp/Pa1651 DNA. The amplified products were sequenced with 454 and Illumina HiSeq platforms. Mapping and de novo assemblies were performed to analyze the quality of data produced from this optimized method.ResultsAnalysis of the sequence data demonstrated that from a single viral plaque of Influenza A, a mapping assembly with 3590-fold average coverage representing 100% of the genome could be produced. The de novo assembled data produced contigs with 30-fold average sequence coverage, representing 96.5% of the genome. Using only 10 pg of starting DNA from bacteriophage F_HA0480sp/Pa1651 in the SISPA protocol resulted in sequencing data that gave a mapping assembly with 3488-fold average sequence coverage, representing 99.9% of the reference and a de novo assembly with 45-fold average sequence coverage, representing 98.1% of the genome.ConclusionsThe optimized SISPA protocol presented here produces amplified product that when sequenced will give high quality data that can be used for de novo assembly. The protocol requires only a single viral plaque or as little as 10 pg of DNA template, which will facilitate rapid identification of viruses during an outbreak and viruses that are difficult to propagate.

Highlights

Whole genome sequencing of viruses and bacteriophages is often hindered because of the need for large quantities of genomic material
The lowest concentration of template required for successful amplification using the modified Sequence Independent Single Primer Amplification (SISPA) protocol was determined by testing a serial dilution of a template phage whose complete genome sequence is known, bacteriophage F_HA0480sp/Pa1651 [GenBank: JN808773.1], which is 37,374 bp in length
Gel-purified SISPA product generated from 10 pg of purified bacteriophage F_HA0480sp/Pa1651 genomic DNA produced a total of 32,415 454 Titanium reads and 5,111,598 HiSeq reads

Summary

Introduction

Whole genome sequencing of viruses and bacteriophages is often hindered because of the need for large quantities of genomic material. A method is described that combines single plaque sequencing with an optimization of Sequence Independent Single Primer Amplification (SISPA). This method can be used for de novo whole genome next-generation sequencing of any cultivable virus without the need for large-scale production of viral stocks or viral purification using centrifugal techniques. Genome sequencing of novel viruses and bacteriophages (phages) is often difficult and time consuming due to the need to grow large-scale, high titer lysates in order to obtain a sufficient quantity of viral nucleic acids for whole genome sequencing.

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