Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing

Abstract

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

Rapid Identification of Drug-Resistant Tuberculosis Genes Using Direct PCR Amplification and Oxford Nanopore Technology Sequencing

“…While WGS is widely used to explore comprehensive genomic information [ 30 , 31 ], in many cases, there are inadequate amounts of clinical specimens for isolating DNA or sequencing. Compared to WGS, amplicon sequencing, which employs PCR products to detect gene information, is an effective and accurate approach for determining genes of known pathogens and has been used for detecting Zika virus [ 32 ], polioviruses [ 33 ], and enteroviruses [ 34 ]. Meanwhile, although high-throughput sequencing offers a faster turnaround time than other detection techniques, purification of bacterial DNA from samples, which typically takes several hours, is a rate-limiting step in the workflow.…”

Acute flaccid myelitis in Switzerland – association with enterovirus D68

“…Sanger sequencing following reverse RNA transcription of a viral genomic region located between the VP4 and the VP2 capsid proteins [19] later identified EV-D68. Shotgun metatranscriptomic sequencing (Illumina® MiSeq, Nextera Flex, 2 × 150 bp, 300 cycles) [20] of the total extracted RNA revealed the presence of few reads (5/4,528,656 reads), which nonetheless unambiguously identified EV-D68 (bootstrap 100%) [21] in two recovered regions (316 bases of 5'UTR, and 132 bases of the capsid protein 1A of VP4; data not shown), covering in total 6.1% of the EV-D68 reference genome sequence AY426531.1. Based on the initial assumption of an immune-mediated inflammatory process, he received high-dose intravenous methylprednisolone, followed by plasmapheresis and intravenous immunoglobulin replacement (table 1).…”

Genome Sequences of Rare Human Enterovirus Genotypes Recovered from Clinical Respiratory Samples in Bern, Switzerland

“…Second, a preparation of 4 μL 5× SuperScript IV reaction buffer (Invitrogen, Carlsbad, CA), 2 μL dithiothreitol (DTT), 200 units SuperScript IV reverse transcriptase (Invitrogen), 40 units RNaseOUT (Invitrogen), and 1 μL NFW was made, and the two preparations were mixed and incubated at 25°C for 10 min, at 50°C for 10 min, and at 80°C for 10 min. The Illumina Nextera DNA Flex kit (paired-end reads, 300 cycles) was used for library preparation, followed by sequencing on a MiSeq benchtop sequencer, as described previously ( 12 ). Read trimming and quality filtering (fastp v0.20.0 [ 13 ]) were followed by genome assembly (SPAdes v3.13.2 [ 14 ]) and multiple sequence alignments (MAFFT v7.313 [ 15 ]).…”