School of Medicine and Health Sciences Poster Presentations

Poster Number

266

Document Type

Poster

Keywords

HTS, quasispecies

Publication Date

4-2017

Abstract

The high level of genetic variability of Human Immunodeficiency Virus type 1 (HIV-1) is caused by the low fidelity of its replication machinery. This leads to evolution of swarm-like viral populations often described as quasispecies. High throughput sequencing (HTS) technology provides higher resolution over Sanger sequencing, enabling detection of low frequency variant genomes. However, quasispecies analysis is still a challenge due to the systematic noise, introduced by HTS technology. This leads to the increase in type I errors (also known as false positives) and the underlying genetic diversity, which can lead to mathematically insolvable type II errors (also known as false negatives). We have developed a pipeline using the tools in the High-performance Integrated Virtual Environment (HIVE), an HTS platform designed for big data analysis and management, to analyze viral populations within each sample and identify their subtype classification and recombination patterns of recombinants. RNA was extracted from 70 plasma samples of chronic HIV-1 infected patients. The 3’ half genomes of HIV-1 were amplified using RT-PCR and PCR products were sequenced using Illumina MiSeq. The paired end reads for each sample were assembled using Geneious software and analyzed for presence of HIV-1 quasispecies using HIVE tools. Subtype analysis of 70 samples using Geneious software identified 17 A1s, 4 Bs, 30 Cs, 1 D, 6 CRF02_AG, and 12 unique recombinant forms (URFs). Additionally, we found up to 178 ambiguous bases in the consensus sequences from 41 viral samples (58.6%), suggesting the presence of viral subpopulations. However, Geneious could not determine the major viral populations in each sample. We analyzed the same HTS reads using the HIV-1 quasispecies analysis pipeline and found one predominant population in 11 samples (15.7 %), two to ten distinct populations in 45 samples (64.3%), 11-20 in 13 samples (18.16%), and 26 in one sample (1.4 %). Interestingly, two equally major viral populations that were not detected by Geneious were identified in five samples (7.1%) by HIVE. The HIV-1 quasispecies analysis pipeline is reliable and more sensitive in its ability to identify distinct viral populations and the recombination patterns not identified by the Geneious software.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Open Access

1

Comments

Poster presented at GW Annual Research Days 2017.

Share

COinS
 

Analysis of HIV-1 quasispecies sequences generated by High Throughput Sequencing (HTS) using HIVE

The high level of genetic variability of Human Immunodeficiency Virus type 1 (HIV-1) is caused by the low fidelity of its replication machinery. This leads to evolution of swarm-like viral populations often described as quasispecies. High throughput sequencing (HTS) technology provides higher resolution over Sanger sequencing, enabling detection of low frequency variant genomes. However, quasispecies analysis is still a challenge due to the systematic noise, introduced by HTS technology. This leads to the increase in type I errors (also known as false positives) and the underlying genetic diversity, which can lead to mathematically insolvable type II errors (also known as false negatives). We have developed a pipeline using the tools in the High-performance Integrated Virtual Environment (HIVE), an HTS platform designed for big data analysis and management, to analyze viral populations within each sample and identify their subtype classification and recombination patterns of recombinants. RNA was extracted from 70 plasma samples of chronic HIV-1 infected patients. The 3’ half genomes of HIV-1 were amplified using RT-PCR and PCR products were sequenced using Illumina MiSeq. The paired end reads for each sample were assembled using Geneious software and analyzed for presence of HIV-1 quasispecies using HIVE tools. Subtype analysis of 70 samples using Geneious software identified 17 A1s, 4 Bs, 30 Cs, 1 D, 6 CRF02_AG, and 12 unique recombinant forms (URFs). Additionally, we found up to 178 ambiguous bases in the consensus sequences from 41 viral samples (58.6%), suggesting the presence of viral subpopulations. However, Geneious could not determine the major viral populations in each sample. We analyzed the same HTS reads using the HIV-1 quasispecies analysis pipeline and found one predominant population in 11 samples (15.7 %), two to ten distinct populations in 45 samples (64.3%), 11-20 in 13 samples (18.16%), and 26 in one sample (1.4 %). Interestingly, two equally major viral populations that were not detected by Geneious were identified in five samples (7.1%) by HIVE. The HIV-1 quasispecies analysis pipeline is reliable and more sensitive in its ability to identify distinct viral populations and the recombination patterns not identified by the Geneious software.

 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.