Universal Target Capture of HIV Sequences From NGS Libraries

Abstract

Background: Global surveillance of viral sequence diversity is needed to keep pace with the constant evolution of HIV. Recent next generation sequencing (NGS) methods have realized the goal of sequencing circulating virus directly from patient specimens. Yet, a simple, universal approach that maximizes sensitivity and sequencing capacity remains elusive. Here we present a novel HIV enrichment strategy to yield near complete genomes from low viral load specimens.Methodology: A non-redundant biotin-labeled probe set (HIV-xGen; n = 652) was synthesized to tile all HIV-1 (groups M, N, O, and P) and HIV-2 (A and B) strains. Illumina Nextera barcoded libraries of either gene-specific or randomly primed cDNA derived from infected plasma were hybridized to probes in a single pool and unbound sequences were washed away. Captured viral cDNA was amplified by Illumina adaptor primers, sequenced on a MiSeq, and NGS reads were demultiplexed for alignment with CLC Bio software.Results: HIV-xGen probes selectively captured and amplified reads spanning the entirety of the HIV phylogenetic tree. HIV sequences clearly present in unenriched libraries of specimens but previously not observed due to high host background levels, insufficient sequencing depth or the extent of multiplexing, were now enriched by >1,000-fold. Thus, xGen selection not only substantially increased the depth of existing sequence, but also extended overall genome coverage by an average of 40%. We characterized 50 new, diverse HIV strains from clinical specimens and demonstrated a viral load cutoff of approximately log 3.5 copies/ml for full length coverage. Genome coverage was <20% for 5/10 samples with viral loads <log 3.5 copies/ml and >90% for 35/40 samples with higher viral loads.Conclusions: Characterization of >20 complete genomes at a time is now possible from a single probe hybridization and MiSeq run. With the versatility to capture all HIV strains and the sensitivity to detect low titer specimens, HIV-xGen will serve as an important tool for monitoring HIV sequence diversity.

Advanced molecular surveillance approaches for characterization of blood borne hepatitis viruses

“…Metagenomic libraries (mNGS) were prepared and quantified essentially as described [13]. Briefly, total nucleic acid was concentrated to 10 μl with RNA Clean and Concentrator-5 spin columns (Zymo Research, CA) and RNA was reverse transcribed with random primers using Superscript III (SSRTIII) 1 st Strand reagents (Life Technologies), followed by 2 nd strand synthesis with Sequenase V2.0 T7 DNA pol (Affymetrix).…”

“…Probe sets were designed essentially as described previously for HIV, with each probe 120 nt in length [13]. Briefly, 60 HBV complete genomes including genotypes A-I were aligned in BioEdit.…”

“…Target enrichment offers an opportunity to significantly boost sensitivity, resulting in improved coverage and higher confidence data [11]. Single stranded DNA probes can be hybridized to reads within mNGS libraries to selectively capture and amplify viral sequences and is particularly useful for samples with low viral loads [12,13]. A post-library capture step (e.g.…”

“…A post-library capture step (e.g. xGen) has successfully been deployed for blood borne RNA viruses in the 10 kb-range length, like HIV and HCV, although it has not yet been evaluated for small viruses like HBV (3.2 kb) and HDV (1.6 kb) [13,14].…”

A comprehensive genomics solution for HIV surveillance and clinical monitoring in a global health setting

“…Second, the design of our probes is unbiased with respect to the range of commonly found HIV viral variants. Abbott Laboratories has recently reported on a similar method, developed in parallel to ours, which they show works across a wider panel of reference genomes [32,33].…”