Abstract
BackgroundWith an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies.Methodology/Principal FindingsWe report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient's infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity.SignificanceThis study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure.
Highlights
The development of a variety of generation sequencing technologies – including Roche/454, Illumina/Solexa, Applied Biosystems SOLiD, Helicos Heliscope, and Pacific Biosciences Single Molecule Real Time (SMRT) sequencing – has made high throughput DNA sequencing possible
The goal of this work is to develop a novel method for amplifying viral RNA into large quantities of DNA suitable for processing into Illumina libraries without relying on polymerase chain reaction (PCR) and primers that assume specific sequences are present within the sample
Numerous studies have previously used 454 generation DNA sequencing to analyze different aspects of human immunodeficiency virus (HIV) biology [13,14,16,17,18,19,28,29,30]. These strong studies did reveal some limitations of the underlying methodology – the use of HIV specific primers that introduced a level of bias into the sequencing data and the difficulty associated with amplifying the entire 10,000 nucleotide HIV genome for analysis
Summary
The development of a variety of generation sequencing technologies – including Roche/454, Illumina/Solexa, Applied Biosystems SOLiD, Helicos Heliscope, and Pacific Biosciences Single Molecule Real Time (SMRT) sequencing – has made high throughput DNA sequencing possible Each of these generation sequencing methods offers differences in read length, the number of reads obtained, and the intrinsic error rate, all of which should be taken into account when choosing a specific sequencing platform. Read length, sequencing error, and the amount of sequence obtained all play a role in the estimation of sequence diversity and error, and methods to account for all of these are constantly evolving We apply both a new nucleic acid amplification and computational analysis that addresses the challenge of aligning generation sequence data to a poorly defined reference genome for characterizing viral quasispecies. With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies
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