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

Stephanie Goya,Gabriel L Rojo,Mariana Viegas,Estefania Tittarelli,Jonathan J Zaiat,Marcelo A Marti,Laura E Valinotto,Alexander L Greninger,Mercedes S Nabaes Jodar,Alicia S Mistchenko

doi:10.1371/journal.pone.0199714

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.

Highlights

Complete genome sequences are a powerful tool for pathogen characterization, molecular surveillance, diagnosis, viral attenuation, response to drug treatment, response to host immune pressure and even new pathogen discovery [1,2,3,4,5]
We evaluated different sample treatments in order to enrich the viral RNA extracted from the respiratory clinical samples prior next generation sequencing (NGS) library preparation
Clinical samples were nasopharyngeal aspirates (NPA) which resulted positive for human respiratory syncytial virus (HRSV) by an indirect immunofluorescence assay as a standard care protocol for respiratory virus routine diagnosis [20]

Summary

Introduction

Complete genome sequences are a powerful tool for pathogen characterization, molecular surveillance, diagnosis, viral attenuation, response to drug treatment, response to host immune pressure and even new pathogen discovery [1,2,3,4,5]. The strategy of NGS includes a wet lab phase in which the target virus can be enriched and a dry lab phase in which an accurate data analysis pipeline should be developed. The enrichment of the target virus is often carried out by viral culture or with PCR amplification by using specific primers [9,10,11,12]. There are many publications which include methodologies to obtain complete viral genomes trying to avoid bias sources but there are not comparative evaluations among them [13,14,15,16,17,18]. It would be desirable that the number of samples to be sequenced be as large as possible, even more when molecular surveillance studies are carried out

Methods

Results

Discussion

Conclusion