Vy-PER: eliminating false positive detection of virus integration events in next generation sequencing data

Abstract

Several pathogenic viruses such as hepatitis B and human immunodeficiency viruses may integrate into the host genome. These virus/host integrations are detectable using paired-end next generation sequencing. However, the low number of expected true virus integrations may be difficult to distinguish from the noise of many false positive candidates. Here, we propose a novel filtering approach that increases specificity without compromising sensitivity for virus/host chimera detection. Our detection pipeline termed Vy-PER (Virus integration detection bY Paired End Reads) outperforms existing similar tools in speed and accuracy. We analysed whole genome data from childhood acute lymphoblastic leukemia (ALL), which is characterised by genomic rearrangements and usually associated with radiation exposure. This analysis was motivated by the recently reported virus integrations at genomic rearrangement sites and association with chromosomal instability in liver cancer. However, as expected, our analysis of 20 tumour and matched germline genomes from ALL patients finds no significant evidence for integrations by known viruses. Nevertheless, our method eliminates 12,800 false positives per genome (80× coverage) and only our method detects singleton human-phiX174-chimeras caused by optical errors of the Illumina HiSeq platform. This high accuracy is useful for detecting low virus integration levels as well as non-integrated viruses.

Figure 1. Detection of virus integrations into the human genome using paired-end sequencing.

After the computationally expensive classical alignment of all paired-end reads to the human genome, the pipeline splits into classical variant calling and virus integration detection.

Figure 2. Vy-PER ideogram summary plot.

Negative example without true virus integrations: Patient genome sequenced with 40× coverage and analysed with highest sensitivity, eliminating 6400 false positives and leaving three unsupported singletons (pseudomonas phage, caviid herpes, cercopithecine herpes) which we manually eliminated as alignment artefacts. PhiX and M13 originated from the 1% Illumina sequencing library control spike-in. The plot shows integration sites down to single chimeras.

Figure 3. Vy-PER ideogram summary plot.

Positive example with true virus integrations: Publicly available whole transcriptome liver cancer data analysed with default sensitivity, showing HBV candidate loci on chromosomes 4, 11 and 16. The plot only shows integrations supported by 10 or more chimeras.

Figure 4. Vy-PER ideogram summary plot.

HBV integration loci into the liver cancer genome detected at low stringency (threshold: 1 chimera), also showing detected phiX singletons.