Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants

Abstract

We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%.Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.

Evolution of Newcastle Disease Virus Quasispecies Diversity and Enhanced Virulence after Passage through Chicken Air Sacs

“…The raw data were treated as described previously with minor refinements (36). Reads obtained from UDPS were demultiplexed corresponding to each sample combination of a multiplex identifier and primer to generate a FASTA file with each sample.…”

Management of direct-acting antiviral agent failures

“…Clonal sequencing is more sensitive, but also more expensive and time-consuming. These limitations are overcome by a technique known as deep sequencing, which allows detection of minor variants present at frequencies as low as 0.5% [70].At present, none of these techniques are commercially available, but a number of laboratories are able to perform sequence analysis of the NS3-4A, NS5A, and NS5Bregions. Nonetheless, the use of these methods in clinical practice is limited, and currently, their interpretation requires expert guidance.…”

Analysis of the Evolution and Structure of a Complex Intrahost Viral Population in Chronic Hepatitis C Virus Mapped by Ultradeep Pyrosequencing

“…As such, we cannot ignore the possibility that in vitro recombination artifacts may also be present in the final data set. The acceptance of low-frequency recombinants requires critical evaluation weighed against the documented preva-lence and characteristics of in vitro artifacts (28,(48)(49)(50). To that end, we submit four main arguments in support of our broader recombination findings.…”