Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Abstract

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection

“…High-throughput sequencing (HTS) is a non-specific technique with the potential to detect both known and unknown adventitious agents including viruses 7,[14][15][16][17] . High-throughput molecular biology methods (HTS combined with a pan-viral microarray) had succeeded in detecting the contamination of Rotarix vaccine by a porcine circovirus 18 .…”

Applying next-generation sequencing to unravel the mutational landscape in viral quasispecies

“…Following RNA extraction and viral RNA enrichment from the host RNA (Forth and Hoper, 2019;Houldcroft et al, 2017;Sathiamoorthy et al, 2018;Singanallur et al, 2019), the first critical quality control (QC) step (Fig. 2) is to test both quantity and integrity of the starting viral RNA (Hauck et al, 2018;Ng et al, 2018;Yang et al, 2016). The necessity to control virus tire or genome copy numbers in comparative studies has been demonstrated in several studies as false positive variant calls become more evident with lower material inputs (Gallet et al, 2017;Illingworth et al, 2017;McCrone and Lauring, 2016).…”

Considerations for Optimization of High-Throughput Sequencing Bioinformatics Pipelines for Virus Detection

“…High background from cellular nucleic acids will reduce the sensitivity of HTS virus detection [ 13 , 14 ]. In most cases, the cellular nucleic acids are the most crucial factor for influencing the sensitivity of virus detection, since they typically dominate the viral read number [ 11 , 15 , 16 ]. Reducing the cellular nucleic acid, such as by nuclease treatment prior to sequencing, may enhance the sensitivity of virus detection in downstream bioinformatics analysis.…”

“…As shown in Figure 1 (closed circles), without incorporation of steps to reduce the background, even an entire Illumina HiSeq flow cell run, generating 2.5 × 10 9 reads in a paired-end mode (for example 2 × 100 bp) would not likely produce a single PCV read below a sensitivity of 1 × 10 4 genomes/mL. While mechanisms of target enrichment and background removal are beyond the scope of this paper [ 11 ], when we explore the effect of improving this signal-to-noise ratio (S/N) by 1000× ( Figure 1 , closed squares), similar levels of sensitivity could then be obtained using a more modest platform (MiSeq, ~1.2 × 10 7 paired-end reads).…”

“…This paper presents our current thinking on bioinformatics pipelines with focus on the choices made in the analysis pipeline—including, but not limited to, sequences, aligners, databases, assembly, and how unmapped reads are managed. Discussions on sample selection and preparation, as well as virus spiking to determine sensitivity, are presented in the context of their influence on the pipeline analysis and details are discussed in another paper from AVDTIG members [ 11 ].…”