HIV-1 genotypic resistance testing using the Vela automated next-generation sequencing platform
Stéphanie RaymondFlorence NicotRomain CarcenacCaroline LefèbvreNicolas JeanneKarine SaunéPierre DelobelJacques Izopet
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To evaluate the diagnostic performance of the Vela next-generation sequencing (NGS) system in conjunction with the Sentosa SQ HIV Genotyping Assay for genotyping HIV-1. Plasma RNA was extracted and templates prepared with the Sentosa SX instrument before sequencing the HIV-1 polymerase on the Sentosa SQ301 Sequencer (PGM IonTorrent). The Vela NGS System was compared with direct sequencing and the 454 GS-FLX (Roche) and MiSeq (Illumina) systems for genotypic resistance testing on clinical samples. The Vela NGS system detected majority resistance mutations in subtype B and CRF02-AG samples at 500 copies/mL and minority variants with a sensitivity of 5% at 100 000 copies/mL. The Vela NGS system and direct sequencing identified resistance mutations with 97% concordance in 46 clinical samples. Vela identified 1/20 of the 1%–5% mutations identified by 454, 5/12 of the 5%–20% mutations and 60/61 of the >20% mutations. Vela identified 3/14 of the 1%–5% mutations identified by MiSeq, 0/2 of the 5%–20% mutations and 47/47 of the >20% mutations. The resistance mutation quantifications by Vela and 454 were concordant (bias: 2.31%), as were those by Vela and MiSeq (bias: 1.06%). The Vela NGS system provides automated nucleic acid extraction, PCR reagent distribution, library preparation and bioinformatics analysis. The analytical performance was very good when compared with direct sequencing, but was less sensitive than two other NGS platforms for detecting minority variants.Keywords:
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To our knowledge, there are no published data on hepatitis C virus (HCV) genotypes in Angola. This study aimed at assessing the distribution of HCV genotypes in seropositive hemodialysis patients in Luanda. Among 51 HCV‐positive subjects included, viremia was detected in 27 (53%). HCV genotyping was performed by bidirectional sequencing of the 5'‐untranslated region by the Sanger method. HCV genotype 4 was largely predominant (20 cases; 74%), followed by genotypes 1b (5 cases; 18.5%), 1a and 2 (one case each; 3.7%). These results suggest that the distribution of HCV genotypes in Angola is similar to that reported from other Central African countries.
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The HCV genotype can be determined by PCR using nested primers to structural or non-structural HCV regions, followed by hybridization analysis of the amplified products. In this study, two different systems, both based on PCR and hybridization analysis, were used to determine HCV genotype in 32 HCV positive patients at the Clinic of Infectious Diseases, University of Chieti. The main difference between these commercially available systems lies in the different PCR target. Amplification of PCR targets was obtained from all samples. Hybridization analysis gave unequivocal results for all samples with both methods, yielding a 100% rate of genotype determination, with a complete correlation at the genotype level. A lower concordance at subtype level (65% concordance) was found, due only to two types of discrepancies. Both methods proved easy to use in our hands, adding evidence to their potential usefulness and reliability in clinical settings.
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Background: Different tests for human papillomavirus (HPV) screening are commercially available, detecting high-risk oncogenic HPV types with a pool of genotype-specific probes.Objectives: The purpose of this study is to compare two commercial methods for HPV genotyping: Linear Arrays HPV genotyping test (Roche), capable of detecting and genotyping 37 different HPV types simultaneously, and the new INNO-LiPA HPV Genotyping Extra (Innogenetics), that detects 28 genotypes, including now HPV-26 (considered probable high-risk genotype) and HPV-82 (considered high-risk genotype), that were not included in the previous version. Both methods also include controls for cell adequacy. Materials and Methods: A total of 100 HPV DNA-positive cervical samples by hybrid capture method were genotyped.Results: Multiple genotypes were found more frequently with Linear Arrays (2.2) than INNO-LiPA (1.7). Comparison analysis was limited to HPV genotypes common to both assays. There were concordant results (absolute agreement between assays) in 65 samples and compatible results (correspondence for some but not all genotypes) were found in 33 samples. In 21 samples additional types by Linear Arrays were detected. In13 samples additional types by INNO-LiPA were detected (in one sample there was additional type detected by Linear Arrays and INNO-LiPA method). Only two samples were considered as discordant (did not show any similarity between the tests) and these were negative by INNO-LiPA and positive by Linear Array (both HPV-73). Analyzing kappa values we have found excellent concordance for 6 genotypes (26, 35, 45, 58, 68, and 70) and very good for other 8 genotypes (6, 16, 31, 33, 51, 52, 53 and 66), Concordance was considered good (0.6-0.8) for 5 genotypes (18, 39, 40, 54 and 56), moderate value (0.4-0.6) for HPV-59 and weak agreement (0.4-0.2) for HPV-11.Conclusions: Both genotyping methods are highly comparable and suitable for clinical and epidemiological studies, as they are partially automated and detect all HPV genotypes of clinical interest.
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Abstract Systematic studies of the circulation of hepatitis C virus (HCV) recombinants in different parts of the world have been initiated only recently, and no detailed information on this subject is available. The aim of the current investigation was to determine the frequency of HCV recombinants in intravenous drug users (IVDU) from two European countries. HCV RNA from serum samples was tested by RT‐PCR with primers derived from the core and NS5B regions with subsequent sequencing and genotype assignment. The 118 samples from Germany (100%) and 45 out of 47 (96%) sera from Russia demonstrated concordant genotyping results. In the two genotype discrepant sera from Russia 2k/1b recombinants were identified. In order to test the hypothesis that the individuals from the IVDU group might be multiply exposed to various genotypes, 145 out of 165 genotyped serum samples, which were found to be positive for anti‐NS4 antibodies, were serotyped with the Murex HCV serotyping kit that is based on detection of antibodies to type‐specific peptides derived from the NS4 proteins of different HCV genotypes. Discrepancy in genotype and serotype attributions was observed in 11% cases. Retesting of 99 type 1a or 3a samples with a set of type‐ and subtype‐specific primers revealed the presence of a mixed infection only in one case (1a/3a). Thus, the cases of the mixed infection with different HCV genotypes as well as the recombinant forms of HCV are very rare even in such a highly exposed group as IVDU. J. Med. Virol. 82:232–238, 2010. © 2009 Wiley‐Liss, Inc.
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Abstract Background: Hepatitis C virus (HCV) genotype is a predictive marker for treatment response. We sequentially evaluated the performances of two nucleic acid amplification tests (NAATs) and one serology assay for HCV genotype: Abbott RealTi me genotype II (RealTi me II), GeneMatrix restriction fragment mass polymorphism (RFMP), and Sysmex HISCL HCV Gr (HISCL Gr). Methods: We examined 281 clinical samples with three assays. The accuracy was assessed using the HCV Genotype Performance Panel PHW204 (SeraCare Life Sciences) for two NAATs. Discrepant cases were re-genotyped by the Versant HCV v.2.0 (line probe 2.0) assay. Results: With the RealTi me II assay, clinic samples were analyzed as follows: genotypes 1b (43.1%), 2 (40.2%), 1 subtypes other than 1a and 1b (12.5%), 3 (1.8%), 4 (1.4%), 1a (0.7%), 6 (0.4%), and mixed (1.1%). The RealTi me II and RFMP assays showed a type concordance rate of 97.5% (274/281) (κ=0.80) and no significant discordance (p=0.25). Both assays accurately genotyped all samples in the Performance Panel by the subtype level. The HISCL Gr assay showed concordance rates of about 91% (κ<0.40) and statistically significant discordances with two NAATs (p<0.05). In confirmation tests, the results of RFMP assay were the most consistent with those of Versant 2.0 assay. Conclusions: The three HCV assays provided genotyping and serotyping results with good concordance rates. The two NAATs (RealTi me II and RFMP) showed comparable performance and good agreement. However, the results of the HISCL Gr assay showed statistically significant differences with those of the NAATs.
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Abstract Genotypes generated from common single nucleotide polymorphisms (SNP) chips have been the sole source of genomic information for current beef cattle genetic evaluations. More recently, the development of low-pass whole-genome sequencing (LPS) followed by imputation to full sequence and variant calls has provided an alternative option to generate genotypes for inclusion into genetic evaluations. The objective of this study was to estimate the genotype concordance between SNP chip and LPS genotypes. Ear samples, via tissue sampling units, were obtained from 226 individuals. A subset of 109 individuals also had blood samples collected. Samples were sent to GeneSeek (Neogen company, Lincoln, NE) for genotyping (100K or 50K) and LPS and imputation was performed by Gencove. A total of 33,451 and 78,032 SNP overlapping between SNP panel and LPS for ear and blood samples, respectively, were used for further analyses. Genotype concordance was performed by counting the number of matching genotypes per SNP between (1) SNP chip vs. LPS in ear and blood samples, and (2) LPS in blood vs. LPS in ear samples. The average concordances between SNP chip vs. LPS in ear and blood samples were 0.97±0.04 and 0.98±0.06, respectively. The average concordance between LPS in blood vs. ear samples was 0.97±0.03. In conclusion, the concordance was high regardless of the tissue type, and, at least in these data, differences in resulting genetic predictions would not be expected between the use of genotypes derived from SNP chips or LPS. Additional work is warranted to determine sensitivity to relationships between target animals and the reference set used for imputation. The USDA is an equal opportunity provider and employer.
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