Challenges in the analysis of viral metagenomes

Rose, Rebecca; Constantinides, Bede; Tapinos, Avraam; Robertson, David L; Prosperi, Mattia

There are several sequencing technologies in widespread use that are capable of reading hundreds of thousands to billions of DNA sequences per run (Reuter, Spacek, and Snyder 2015) . The current market leader, Illumina, manufactures instruments capable of generating billions of 150 base pair (bp) paired end reads (see 'Glossary') per run, with read lengths of up to 300 bp. The Illumina short read platform is widely used for analyses of viral genomes and metagenomes, and, given sufficient sequencing coverage, enables sensitive characterization of lowfrequency variation within viral populations (e.g. HIV resistance mutations as low as 0.1% (Li et al. 2014) ). Ion Torrent (ThermoFisher) is capable of generating longer reads than Illumina at the expense of reduced throughput and a higher rate of insertion and deletion (indel) error (Eid et al. 2009 ). Single molecule real-time sequencing commercialized by Pacific Biosciences (PacBio) produces much longer (>10 kbp) reads from a single molecule without clonal amplification, which eliminates the errors introduced in this step. However, this platform has a high (10%) intrinsic error rate, and remains much more expensive than Illumina sequencing for equivalent throughput. The Nanopore platform from Oxford Nanopore Technologies, which includes the pocket sized MinION sequencer, also implements long read single molecule sequencing, and permits truly real-time analysis of individual sequences as they are generated. Although more affordable than PacBio single molecule sequencing, the Nanopore platform also suffers from high error rates in comparison with Illumina (Reuter, Spacek, and Snyder 2015) . However, the technology is maturing rapidly and has already demonstrated potential to revolutionize pathogen surveillance and discovery in the field, as well as enabling contiguous assembly of entire bacterial genomes at relatively low cost (Feng et al. 2015; Quick et al. 2015; Hoenen et al. 2016 ). Hybrid sequencing strategies using both long and short reads leverage the ability of long reads to resolve repetitive DNA regions while benefitting from the high accuracy of short reads, at the expense of additional sequencing, library preparation and data analysis (Madoui et al. 2015) .