Sequencing viral genomes from a single isolated plaque

Abstract

Background: 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.

Methods: A 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.

Results: Analysis 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.

Conclusions: The 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.

Figure 1

Mapping and de novo assembly coverage sequencing results for the phage SISPA product from 10 pg of genomic DNA. The black lines along the x-axis are the de novo assembled contigs mapped to the reference (light gray line across the x-axis). Coverage for the de novo contigs is shown with dark gray bars in the graph (left y-axis). The coverage from mapping the reads to the reference is represented with a black line (right y-axis).

Figure 2

Mapping and de novo assembly coverage sequencing results for the flu SISPA product from a single plaque. The black lines along the x-axis are the de novo assembled contigs mapped to the reference (light gray line across the x-axis). Coverage for the de novo contigs is shown with dark gray bars in the graph (left y-axis). The coverage from mapping the reads to the reference is represented with a black line (right y-axis).