Advanced

Low-Bias RNA Sequencing of the HIV-2 Genome from Blood Plasma

James, Katherine L.; de Silva, Thushan I.; Brown, Katherine; Whittle, Hilton; Taylor, Stephen; McVean, Gilean; Esbjörnsson, Joakim LU and Rowland-Jones, Sarah L. (2019) In Journal of Virology 93(1).
Abstract

Accurate determination of the genetic diversity present in the HIV quasispecies is critical for the development of a preventative vaccine: in particular, little is known about viral genetic diversity for the second type of HIV, HIV-2. A better understanding of HIV-2 biology is relevant to the HIV vaccine field because a substantial proportion of infected people experience long-term viral control, and prior HIV-2 infection has been associated with slower HIV-1 disease progression in coinfected subjects. The majority of traditional and next-generation sequencing methods have relied on target amplification prior to sequencing, introducing biases that may obscure the true signals of diversity in the viral population. Additionally, target... (More)

Accurate determination of the genetic diversity present in the HIV quasispecies is critical for the development of a preventative vaccine: in particular, little is known about viral genetic diversity for the second type of HIV, HIV-2. A better understanding of HIV-2 biology is relevant to the HIV vaccine field because a substantial proportion of infected people experience long-term viral control, and prior HIV-2 infection has been associated with slower HIV-1 disease progression in coinfected subjects. The majority of traditional and next-generation sequencing methods have relied on target amplification prior to sequencing, introducing biases that may obscure the true signals of diversity in the viral population. Additionally, target enrichment through PCR requires a priori sequence knowledge, which is lacking for HIV-2. Therefore, a target enrichment free method of library preparation would be valuable for the field. We applied an RNA shotgun sequencing (RNA-Seq) method without PCR amplification to cultured viral stocks and patient plasma samples from HIV-2-infected individuals. Libraries generated from total plasma RNA were analyzed with a two-step pipeline: (i) de novo genome assembly, followed by (ii) read remapping. By this approach, whole-genome sequences were generated with a 28× to 67× mean depth of coverage. Assembled reads showed a low level of GC bias, and comparison of the genome diversities at the intrahost level showed low diversity in the accessory gene vpx in all patients. Our study demonstrates that RNA-Seq is a feasible full-genome de novo sequencing method for blood plasma samples collected from HIV-2-infected individuals.IMPORTANCE An accurate picture of viral genetic diversity is critical for the development of a globally effective HIV vaccine. However, sequencing strategies are often complicated by target enrichment prior to sequencing, introducing biases that can distort variant frequencies, which are not easily corrected for in downstream analyses. Additionally, detailed a priori sequence knowledge is needed to inform robust primer design when employing PCR amplification, a factor that is often lacking when working with tropical diseases localized in developing countries. Previous work has demonstrated that direct RNA shotgun sequencing (RNA-Seq) can be used to circumvent these issues for hepatitis C virus (HCV) and norovirus. We applied RNA-Seq to total RNA extracted from HIV-2 blood plasma samples, demonstrating the applicability of this technique to HIV-2 and allowing us to generate a dynamic picture of genetic diversity over the whole genome of HIV-2 in the context of low-bias sequencing.

(Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
HIV-2, next-generation sequencing, RNA sequencing, vpx, whole genome
in
Journal of Virology
volume
93
issue
1
publisher
American Society for Microbiology
external identifiers
  • scopus:85058444403
ISSN
1098-5514
DOI
10.1128/JVI.00677-18
language
English
LU publication?
yes
id
b09e13b4-4da3-4685-b9c9-f2314a881589
date added to LUP
2019-01-03 08:28:30
date last changed
2019-02-20 11:41:21
@article{b09e13b4-4da3-4685-b9c9-f2314a881589,
  abstract     = {<p>Accurate determination of the genetic diversity present in the HIV quasispecies is critical for the development of a preventative vaccine: in particular, little is known about viral genetic diversity for the second type of HIV, HIV-2. A better understanding of HIV-2 biology is relevant to the HIV vaccine field because a substantial proportion of infected people experience long-term viral control, and prior HIV-2 infection has been associated with slower HIV-1 disease progression in coinfected subjects. The majority of traditional and next-generation sequencing methods have relied on target amplification prior to sequencing, introducing biases that may obscure the true signals of diversity in the viral population. Additionally, target enrichment through PCR requires a priori sequence knowledge, which is lacking for HIV-2. Therefore, a target enrichment free method of library preparation would be valuable for the field. We applied an RNA shotgun sequencing (RNA-Seq) method without PCR amplification to cultured viral stocks and patient plasma samples from HIV-2-infected individuals. Libraries generated from total plasma RNA were analyzed with a two-step pipeline: (i) de novo genome assembly, followed by (ii) read remapping. By this approach, whole-genome sequences were generated with a 28× to 67× mean depth of coverage. Assembled reads showed a low level of GC bias, and comparison of the genome diversities at the intrahost level showed low diversity in the accessory gene vpx in all patients. Our study demonstrates that RNA-Seq is a feasible full-genome de novo sequencing method for blood plasma samples collected from HIV-2-infected individuals.IMPORTANCE An accurate picture of viral genetic diversity is critical for the development of a globally effective HIV vaccine. However, sequencing strategies are often complicated by target enrichment prior to sequencing, introducing biases that can distort variant frequencies, which are not easily corrected for in downstream analyses. Additionally, detailed a priori sequence knowledge is needed to inform robust primer design when employing PCR amplification, a factor that is often lacking when working with tropical diseases localized in developing countries. Previous work has demonstrated that direct RNA shotgun sequencing (RNA-Seq) can be used to circumvent these issues for hepatitis C virus (HCV) and norovirus. We applied RNA-Seq to total RNA extracted from HIV-2 blood plasma samples, demonstrating the applicability of this technique to HIV-2 and allowing us to generate a dynamic picture of genetic diversity over the whole genome of HIV-2 in the context of low-bias sequencing.</p>},
  articleno    = {e00677-18},
  author       = {James, Katherine L. and de Silva, Thushan I. and Brown, Katherine and Whittle, Hilton and Taylor, Stephen and McVean, Gilean and Esbjörnsson, Joakim and Rowland-Jones, Sarah L.},
  issn         = {1098-5514},
  keyword      = {HIV-2,next-generation sequencing,RNA sequencing,vpx,whole genome},
  language     = {eng},
  number       = {1},
  publisher    = {American Society for Microbiology},
  series       = {Journal of Virology},
  title        = {Low-Bias RNA Sequencing of the HIV-2 Genome from Blood Plasma},
  url          = {http://dx.doi.org/10.1128/JVI.00677-18},
  volume       = {93},
  year         = {2019},
}