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Evaluation of an Ultra-deep Sequencing Method to Identify Minority Sequence Variants in the HIV-1 env Gene from Clinical Samples
Marilyn Lewis*1, I James1, M Braverman2, B Desany2, T Jarvie2, M Penny1, R Harrigan3, M Youle4, R Hernandez1, and E Van Der Ryst1
1Pfizer Global R&D, Sandwich, UK; 2454 Life Sci, Branford, CT, US; 3BC Ctr for Excellence in HIV/AIDS, Vancouver, Canada; and 4Royal Free Hosp, London, UK
Background: The aim of this study was to identify
minority HIV-1 env sequences in a
pre-treatment sample of a patient identified as having only R5 HIV at screening
in whom CXCR4-using virus was detected following 10
days of monotherapy with maraviroc (MVC). The 454-technology simultaneously
sequences hundreds of thousands of clones within a single sample using an
emulsion-based method to amplify and immobilize DNA fragments spanning the gene
of interest. This method should therefore detect individual clones present in a
heterogeneous virus pool at a frequency of <1%.
Methods: Amplicons of the full-length env gene (2.5 kb) from 2 plasma samples
(days 1 and 11) were randomly fragmented and sequenced using a 200-bp average
read length protocol to a depth of >100,000 reads. From the 2 samples the
454-reads were mapped against the HIV-1 sequence database from Los Alamos
National Laboratory (LANL). Using the closest match from the database,
sequencing reads were incorporated into the existing HIV-1 multiple sequence
alignment from LANL. Sequences were assigned a CXCR4-using genotype according
to the 11/25 charge rule.
Results: A total of 104,628 and 191,637 reads were
obtained for the days 1 and 11 samples, respectively, and compared to the LANL env alignment. In this fashion, >99%
of the reads could be mapped. The heterogeneity of env made it difficult to reconstruct full-length quasispecies, so
we focused on reads that fully spanned the V3 region. This resulted in 4104 and
8275 V3 spanning reads for the days 1 and 11 samples, respectively. The day 11
sample contained 20 unique V3 sequences at a frequency of >0.1%, with 90% of
these having a CXCR4-using genotype. In contrast, no CXCR4-using sequence was
identified amongst the 23 unique V3 sequences from day 1, present at >0.1%
frequency. By using the BLAST algorithm and a typical CXCR4-using sequence from
day 11, a single sequence with a CXCR4-using genotype was identified in the day
1 sample (0.02%).
Conclusions: Ultra-deep sequencing provided good-quality
sequences and detected minority HIV-1 env
sequences in a clinical sample. A CXCR4-using genotypic sequence was detected
from the day 1 sample but only following secondary analysis. These results
concur with the phenotypic tropism assessment of these samples.
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