Genomic analysis of serologically untypable human enteroviruses in Taiwan

Abstract

Background Human enteroviruses contain over 100 serotypes. We have routinely conducted enterovirus surveillance in northern Taiwan; but about 10% of isolates could not be serotyped using traditional assays. Next-generation sequencing (NGS) is a powerful tool for genome sequencing. Methods In this study, we established an NGS platform to conduct genome sequencing for the serologically untypable enterovirus isolates. Results Among 130 serologically untypable isolates, 121 (93%) of them were classified into 29 serotypes using CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer)-based RT-PCR to amplify VP1 genes (VP1-CODEHOP). We further selected 52 samples for NGS and identified 59 genome sequences from 51 samples, including 8 samples containing two virus genomes. We also detected 23 genome variants (nucleotide identity < 90% compared with genome sequences in the public domain) which were potential genetic recombination, including 9 inter-serotype recombinants and 14 strains with unknown sources of recombination. Conclusions We successfully integrated VP1-CODEHOP and NGS techniques to conduct genomic analysis of serologically untypable enteroviruses. Electronic supplementary material The online version of this article (10.1186/s12929-019-0541-x) contains supplementary material, which is available to authorized users.

Cost-effective complete genome sequencing using the MinION platform for identification of recombinant enteroviruses

“…To ensure the quality of the reads, a filtering process was applied to retain reads with a length between 1,000 and 8,000 bp, resulting in 13.4 Gbp (83.75%) for further analysis. As shown in Table 1 , 36 enterovirus isolates were sequenced, including 26 isolates from species A, 9 from species B, and 1 isolate from species C. Of these, 18 isolates (P01–P18) had previously been sequenced using the Illumina platform (accession numbers starting with MF, in Table 1 ) ( 2 ). The comparison between the genome sequences obtained from MinION and Illumina platforms revealed exceptionally high average nucleotide identities of 99.98%, as indicated in Table 1 .…”

“…After completing the size-selection process, the sequencing proportion of long reads increased considerably from 45.88% to 68.78%. A total of 70 enterovirus isolates were sequenced in this high-throughput run (Table S2), including 33 samples (H01–H13 and H22–H41) that were previously sequenced using the Illumina platform ( 2 ), and 8 samples (H14–H21) that were sequenced in the pilot run. The average nucleotide identity remained consistently high at 99.98%, both in comparing the corresponding genome sequences obtained between the Illumina and MinION platforms and between the pilot and the high-throughput runs.…”

“…Enteroviruses (EVs) belong to the Picornaviridae family and are widespread and environmentally stable RNA viruses ( 1 ). They are characterized by a conserved genomic structure, consisting of a single-stranded RNA genome ranging in size from 7.2 to 8.5 kb ( 2 ). The capsid proteins, VP1 to VP4, are encoded in the P1 region of the enterovirus genome, whereas the nonstructural proteins are encoded in the remaining P2 and P3 regions ( 3 ).…”

“…However, achieving complete genome sequencing can be challenging due to their high variability and dynamic nature in epidemics. Challenges arise in primer design to ensure coverage of diverse strains and in managing the sequencing costs associated with large-scale genomic studies ( 2 , 17 , 18 ).…”

“…This is primarily attributed to its portability and cost-effectiveness compared to other sequencing technologies. In our previous study ( 2 ), we employed Illumina sequencing technology to sequence the genome of 52 enterovirus isolates. However, the cost associated with constructing genome libraries using this method was not sufficiently low to make it suitable for widespread use in virus surveillance.…”