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Sensitive population profiling and genome assembly of HIV and Flaviviruses using ultra-deep sequencing technologies

  • Matthew R Henn1,
  • Niall J Lennon1,
  • Ruchi Newman1,
  • Patrick Charlebois1,
  • Christian Boutwell2,
  • Molly OhAinle3,
  • Aaron Berlin1,
  • Elizabeth Ryan1,
  • Christine Malboeuf1,
  • Alex Macalalad1,
  • Monica Casali1,
  • Rachel Erlich1,
  • Henry Bigelow1,
  • Lisa Green1,
  • Sante Gnerre1,
  • Sarah Young1,
  • Joshua Levin1,
  • Chad Nusbaum1,
  • Bruce D Walker2,
  • Michael S Diamond4,
  • Laura D Kramer5,
  • Gregory D Ebel6,
  • Eva Harris3,
  • Todd M Allen2 and
  • Bruce W Birren1
Genome Biology201011(Suppl 1):P18

Published: 11 October 2010


Genome AssemblyViral SequencingGenome Sequence DataSensitive IdentificationFull Genome Sequence

Viral diseases such as HIV/AIDS and Dengue have an enormous impact on human health worldwide. Despite this, application of new sequencing technologies to viral genomics has lagged. We are using genome sequence data to study how populations of single stranded RNA viruses, including HIV, Dengue, West Nile and Hepatitis C, evolve within infected individuals in response to host immune, therapeutic and vaccine pressures. To support this, we have developed high-throughput sequencing, assembly and population profiling pipelines based on 454 and Illumina technology that are tuned to the specific needs of viral sequencing. These strategies can capture full genome sequences and can profile sequence diversity at each residue in the genome with unprecedented sensitivity. Our analytical pipeline for 454 and Illumina data (i) generates complete genome assemblies from short sequencing reads derived from populations with high rates of variation; and (ii) detects and quantifies variants in these populations with high sensitivity, while differentiating true variants from process errors.

Initial results with our viral sequencing and analysis pipeline are extremely promising. We detected rare variants to below 1% frequency, revolutionizing our ability to accurately assess the earliest events in viral evolution. We have demonstrated effective assessment of genome-wide diversity during acute HIV infection, enabling rapid, affordable, and highly sensitive identification of the earliest cellular immune responses to HIV. This has allowed us to detect earlier evolutionary events, demonstrating, for example, that HIV cytotoxic T-lymphocyte (CTL) escape can occur much faster than previously known. In addition, we have shown that the extent of intra-host diversity in Flaviviruses such as DENV and WNV is different between these closely related viruses with the latter exhibiting greater genetic diversity. The results presented here demonstrate the power of scalable, next generation sequencing-based methodologies as a genome-wide and unbiased global approach to profiling genomic diversity in intra-host populations of single stranded RNA viruses.



This project has been funded by the NIAID NIH, Dept. HHS, under Contract No. HHSN272200900006C and the Gates Foundation.

Authors’ Affiliations

Broad Institute, Cambridge, USA
Ragon Institute, Boston, USA
University of California, Berkeley, USA
Washington University School of Medicine, St. Louis, USA
Wadsworth Center, Albany, USA
University of New Mexico, Albuquerque, USA


© Henn et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd.