Characterizing HIV Mutations While Lowering NGS Bias (Scientific)

It’s estimated that 38 million human beings are infected with HIV worldwide, using next generation sequencing (NGS) technology to understand the virus mutation and resistance with determination to design successful therapies to treat HIV¹.  Today sequencing is more cost efficient and faster than it was almost 10-15 years ago, now companies have developed diverse next generation sequencers that vary on the read length and error rates.  Still sequencing the HIV virus presents its challenges; library containing large quantity of reference DNA, when large reference sequences aren’t available PCR is utilized which affect analysis of diversity in the sample³.  To sequence large quantities of HIV virus isolated from clinical samples using Illumina NGS, then the amplified nucleic acid is aligned to a poorly defined reference genome in order to characterize entire viral populations².  One of the difficulty of working with HIV it’s the high rate of viral production and error prone mechanism of viral reverse transcriptase that leads to a heterogeneous viral population making it somewhat impossible to sequence globally the HIV virus. 

In Stephanie M. Willerth and teams study, to develop Illumina libraries without relying on PCR and primers a large quantity of viral dsDNA collected from clinical in vitro CD4+T cells was isolated into RNA using QIAamp viral RNA Mini Kit³.  Then the RNA is converted into an extensive ssDNA, and the ssDNA was converted to double strand using NuGEN WT-Ovation³.  The cDNA was processed into an Illumina library using the Illumina Genomic DNA Sample Prep kit³.  To validate the procedure the prepared Illumina library was compared to a homogenous control, HIV stain NLA-3, using MAQ program³.  After computational analysis for insertion and deletions by MAQ the sequence was scanned against Stanford University HIV Drug Resistance Database which identified drug resistant mutations in the clinical samples³. 

The study successfully proves that the HIV genome can be sequenced efficiently and rapidly by eliminating primers there, for lowering the bias of characterizing HIV population while using next generation sequencing.  This method could be applied to study larger HIV populations for mutations and viral resistance to drugs, also the method can assist in providing genomes of organisms that are hard to cultivate.

References:

1.       CDC. HIV.  Retrieved from: http://www.cdc.gov/hiv/library/factsheets/index.html

2.       Daniel Aird, Michael G Ross, Wei-Sheng Chen, Maxwell Danielsson, Timothy Fennell, Carsten Russ, David B Jaffe, Chad Nusbaum and Andreas Gnirke.  2011.  Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.  Genome Biology. 

3.       Stephanie M. Willerth, He ´lder A. M. Pedro, Lior Pachter, Laurent M. Humeau, Adam P. Arkin and David V. Schaffe.  2010.  Development of a Low Bias Method for Characterizing Viral Populations Using Next Generation Sequencing Technology.  PLoSONE.  5(10)