Next-generation sequencing in clinical virology: Discovery of new viruses

Abstract

Viruses are a cause of significant health problem worldwide, especially in the developing nations. Due to different anthropological activities, human populations are exposed to different viral pathogens, many of which emerge as outbreaks. In such situations, discovery of novel viruses is utmost important for deciding prevention and treatment strategies. Since last century, a number of different virus discovery methods, based on cell culture inoculation, sequence-independent PCR have been used for identification of a variety of viruses. However, the recent emergence and commercial availability of next-generation sequencers (NGS) has entirely changed the field of virus discovery. These massively parallel sequencing platforms can sequence a mixture of genetic materials from a very heterogeneous mix, with high sensitivity. Moreover, these platforms work in a sequence-independent manner, making them ideal tools for virus discovery. However, for their application in clinics, sample preparation or enrichment is necessary to detect low abundance virus populations. A number of techniques have also been developed for enrichment or viral nucleic acids. In this manuscript, we review the evolution of sequencing; NGS technologies available today as well as widely used virus enrichment technologies. We also discuss the challenges associated with their applications in the clinical virus discovery.

Keywords: Metagenomics; Next-generation sequencers; PCR; Rolling circle amplification; Sequence-independent single-primer amplification; Virus discovery; Virus discovery based on cDNA-AFLP.

Figure 1

Diagrammatic representation of main steps of clinical virus discovery by next-generation sequencer based technologies.

Figure 2

Different virus nucleic acid enrichment techniques. A: Sequence-independent single-primer amplification. Initially viral RNA and ssDNA is transcribed into complementary DNA (cDNA) using reverse transcriptase (RT) and DNA Pol I respectively, with the help of tagged-primers having defined sequence at the 5’ end while random nucleotides at the 3’ end. Subsequently, second strand synthesis is performed using DNA Pol I (Klenow) to make the cDNA double stranded (dsDNA). Now all the nuceic acids present in the reaction are dsDNA fragments have tagged sequence at their ends. Finally, anchored dsDNA is amplified with primers annealing to the adapter specific sequences, PCR product are checked and ready for analysis through cloning-sequencing or direct sequencing through next-generation sequencers (NGS); B: Virus discovery based on cDNA-AFLP. Initially viral RNA is reverse transcribed into complementary DNA (cDNA) using RT and random primers. Subsequently, second strand synthesis is performed using DNA Pol I (Klenow) to make the cDNA double stranded (dsDNA). In this step, other viral single stranded DNA (ssDNA) viral is also converted to dsDNA. Now all the nuceic acids present in the reaction are dsDNA. In the next step dsDNA are digested with a set of frequent cutter restriction endonucleases, which produce asymmetric cuts. Now specially designed matching anchor-adapters are ligated ends of the restriction fragments using DNA Ligase. Finally, anchored dsDNA is amplified with primers annealing to the adapter specific sequences, PCR product are checked and ready for analysis through cloning-sequencing or direct sequencing through NGS; C: Rolling circle amplification. Amplification of multiply primed single stranded circular viral genomes. 3’-exonuclease resistant primers randomly bind the genome and are elongated by the Phi29 polymerase. The growing strand subsequently displaces the preceding strand of the DNA, making the strand available for binding of random primers and further elongation. This cyclic displacement and elongation leads to a highly branched structure of growing DNA, which is linear in topology. Rolling circle amplification has the capability to specifically enrich the circular ssDNA genomes in an environment of other genetic materials, and could then be characterized by NGS.