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An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

ABSTRACT: Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ? E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1-3 (HPIV1-3) were also obtained with the selected optimal methodology.

SUBMITTER: Goya S

PROVIDER: S-EPMC6016902 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

Goya Stephanie S Valinotto Laura E LE Tittarelli Estefania E Rojo Gabriel L GL Nabaes Jodar Mercedes S MS Greninger Alexander L AL Zaiat Jonathan J JJ Marti Marcelo A MA Mistchenko Alicia S AS Viegas Mariana M

PloS one 20180625 6

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain ...[more]

PMID: 29940028

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Project description:RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Project description:The human respiratory syncytial virus (RSV) is considered one of the most common viruses that infect children globally. The virus is known to have extensive gene sequence variability within and between RSV groups A and B globally; however, there is no information on the whole-genome characterization and diversity of RSV in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with acute respiratory tract infection (ARTI) in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with ARTI in Kuwait. Between January 2020 and September 2022, 7,093 respiratory samples were collected from hospitalized infants, children, and adults and were analyzed for respiratory viruses by multiplex real-time PCR. Whole-genome sequencing using the Oxford Nanopore sequencing technology was performed on 84 RSV-positive samples. The results revealed a higher prevalence of group A (76%) than group B (24%) RSV isolates. Phylogenetic analysis showed that RSV-A strains clustered with the GA2.3.5 sub-genotype and RSV-B strains clustered with the GB5.0.5a sub-genotype; however, forming new lineages of RSV-A and RSV-B circulated in Kuwait during this period. Genetic variability was higher among the group A viruses than group B viruses, and the rate of synonymous and missense mutations was high in genes other than the G protein-coding gene. We also detected several known and unique molecular markers in different protein-coding genes. This is the first study in Kuwait to characterize the whole genomes of RSV A and B to identify the circulating genotypes, comprehend the genetic diversity and the evolution of the virus, and identify important genetic markers associated with specific genotypes.IMPORTANCEWhole-genome sequencing of respiratory syncytial virus (RSV) strains in Kuwait using MinION Nanopore technology was used to characterize and analyze the genotypes and sub-genotypes of the RSV circulating among patients with acute respiratory tract infections in Kuwait. This study also identified known and unknown gene mutations and imported genetic markers associated with specific genotypes. These results will assist in establishing a framework for RSV classification and allow for a better consideration of the mechanisms leading to the generation of diversity of RSV. In addition, these data will allow a comparison of vaccine viruses with those in Kuwait, providing useful insights into future vaccine and therapy strategies for RSV in Kuwait.

Dataset Information

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

Publications

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

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