A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing

Abstract

Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.

Figure 1. NGS of HCV biopsy RNA.

Extracted and pooled (×6) HCV infected biopsy RNA was reverse transcribed and amplified prior to Illumina NGS and mapped to the HCV reference 3a.NZL. NC_0009824 (Los Alamos HCV database).

Figure 2. Enrichment methodologies.

a: Illustration of the key steps in isolating and enriching viral nucleic acids relative to host nucleic acids prior to sequencing by NGS. Liver tissue cells were broken in an isotonic buffer supplemented with BSA using a hard tissue probe and an Omni-Homogeniser with a dry-ice freeze/thaw step (repeated 3×). To ensure that cells membranes were broken the lysate was checked microscopically. b: Comparison of Alumina/pestle-grinding against hard-tissue probe homogenisation with freeze/thaw cycles (+/− the addition of Benzonase). (2 mm³ fragments were dissected from an HCV infected liver sample. Duplicate tissue samples were used for each protocol. HCV nucleic acids were measured in triplicate by dual labelled qPCR assay with NIBSC standard.

Figure 3. Quantitative PCR (viral genome copy number in liver samples).

A SYBR-green assay and plasmid standard was used for the CAV and CPV samples, whilst the HCV and HBV assays used dual labelled probed with NIBSC standards. Total RNA extracted for HCV and Total DNA extracted for HBV, CPV and CAV. The Genomic strand copy number for HCV was estimated by performing the RT step in the presence of the reverse primer only.

Figure 4. Quantitative PCR (viral copy number estimations of prepared samples for NGS).

For each viral liver sample, 2 mm³ of liver was used separately for total RNA extraction, total DNA extraction, DNA viral enrichment and RNA viral enrichment. qPCR assays were performed using 20 ng of each sample (estimated by Pico-green assay) as an input in triplicate. The samples subsequently mass sequenced are indicated with an asterisk.

Figure 5. NGS virus sequence mapping to reference genomes.

To assess the relative difference between total RNA and total DNA extracts with the virally enriched samples, the NGS viral reads were mapped to respective reference genomes.

Figure 6. Virus reference reads as percentage of NGS datasets.

Viral reference sequencing reads shown as a percentage of total total reads from the Illumina NGS datasets for the differentially processed liver samples.

Figure 7. De novo assembly of viral contigs.

NGS reads for infected liver samples and their processed fractions, were used to generate contigs using ABySS and CLC de novo assembly algorithms. Contigs (length over 200 nt) were mapped to the viral reference genomes with the length and coverage indicated (solid black lines). Regions of the reference genomes not covered by the contigs are indicated with dashed lines.