Targeted deep sequencing of HIV-1 using the IonTorrentPGM platform

Abstract

Summary The characterization of mixed HIV-1 populations is a key question in clinical and basic research settings. This can be achieved through targeted deep sequencing (TDS), where next-generation sequencing is used to examine in depth a sub-genomic region of interest. This study explores the suitability of IonTorrent PGM(LifeTechnologies) for the TDS-based analysis of HIV-1 evolution. Using laboratory reagents and primary specimens sampled at pre-peak viremia the error rates from misincorporation and in vitro recombination were<0.5%. The sequencing error rate was 2- to 3-fold higher in/around homopolymeric tracts, and could be discerned from true polymorphism using bidirectional sequencing. The limit of detection of complex variants was further lowered by using haplotyping. The application of this system was illustrated on primary samples from an individual infected with HIV-1 followed from pre-peak viremia through six months post-acquisition. TDS provided an augmented view of the extent of genetic diversity, the covariation among polymorphisms, the evolutionary pathways, and the boundaries of the mutational space explored by the viral swarm. Based on its performance, the system can be applied for the characterization of minor viral variants in support of studies of viral evolution, which can inform the rational design of the next generation of vaccines and therapeutics.

Next generation sequencing improves detection of drug resistance mutations in infants after PMTCT failure

“…Similar to previous studies and considering read coverage (defined as the number of reads with adequate quality for RT amino acid positions 1–230), a threshold of 0.5% was used for reporting minor variants [14]. Median read-coverage was greater with MiSeq: 74 181 (IQR: 50 173–104 089) than with PGM 8 939 (IQR: 4 521–12 585) resulting in an overall higher coverage per template (Table 2).…”

“…Next generation sequencing (NGS) offers an attractive alternative to potentially detect all DRMs across the HIV- 1 reverse transcriptase (RT) coding region. The read lengths of modern NGS systems including Roche 454 (454 Life Sciences, Branford, CT, USA), Ion Torrent Personal Genome Machine (PGM) (Life Technologies, Carlsbad, CA, USA) (PGM) and MiSeq (Illumina, San Diego, CA, USA), also permit the study of linkage between some DRMs [14]. A recent study found good correlation of Roche 454 sequencing for K103N and Y181C, when screening PMTCT exposed children (less than 2 years of age) prior to cART initiation[15].…”

Immunological and virological characteristics of human immunodeficiency virus type 1 superinfection: implications in vaccine design

“…The next-generation sequencing (NGS) method is very sensitive for the detection of minority variants, as low as 0.25% of the population in samples. The use of sequence identification tags has significantly reduced the cost and increased the throughput of the NGS method [44–46]. Thus, it has been widely used to detect superinfection in a large number of samples by analyzing multiple genome regions to determine the incidence of superinfection [47].…”

Triple Class HIV-1 Drug Resistance in Croatia: the First Report

“…To achieve the subsequent long-term treatment success, the resistance must be held under control by monitoring in both routine diagnostic setting and clinical research 1,9 . Sanger sequencing has been the golden standard for characterization of HIV resistance so far, but with the development of new ˝deep sequencing˝ technologies and their increased sensitivity for detection of minor mutations, many clinical laboratories and research groups begun to implement it in their research [10][11][12][13][14] . With Sanger sequencing it is possible to detect viral quasispecies present in 15-20% of the total viral population while viral variants present in lower frequency will not be detected.…”

“…On the other hand, deep sequencing allows analysis of viral minor variants represented in <1% of the total population, which provides a new insight on pathogenesis of HIV-1 infection. The advantages of such sequencing are of exceptional importance because deep sequencing can help monitor the resistance while minor resistant variants are still in development and do not dominated the viral population [10][11][12][13][14] . The following groups of antiretroviral drugs are used for treatment of HIV-infection: nucleoside (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) and integrase inhibitors (INSTI).…”