HCV Genotyping with Concurrent Profiling of Resistance-Associated Variants by NGS Analysis

Determination of viral characteristics including genotype (GT), subtype (ST) and resistance-associated variants (RAVs) profile is important in assigning direct-acting antivirals regimes in HCV patients. To help achieve the best clinical management of HCV patients, a routine diagnostic laboratory should aim at reporting accurate viral GT/ST and RAVs using a reliable diagnostic platform of choice. A laboratory study was conducted to evaluate performance characteristics of a new commercial next-generation sequencing (NGS)-based HCV genotyping assay in comparison to another widely used commercial line probe assay for HCV genotyping. Information on RAVs from deeply sequenced NS3, NS5A and NS5B regions in samples classified as HCV 1a and 1b was harnessed from the fully automated software. Perfect (100%) concordance at HCV genotype level was achieved in GT2 (N = 13), GT3 (N = 55) and GT5 (N = 7). NGS refined the ST assignment in GTs 1, 4 and 6, and resolved previously indeterminate GTs reported by line probe assay. NGS was found to have consistent intra- and inter-run reproducibility in terms of genotyping, subtyping and RAVs identification. Detection of infections with multiple HCV GTs or STs is feasible by NGS. Deep sequencing allows sensitive identification of RAVs in the GT 1a and 1b NS3, NS5A and NS5B regions, but the list of target RAVs is not exhaustive

Prevalence of human endogenous retroviral element associates with Hodgkin's lymphoma incidence rates

Human endogenous retrovirus-H (HERV-H) is implicated in leukaemias and lymphomas, but the precise molecular mechanism underlying HERV-mediated carcinogenesis remains unknown. We determined the prevalence of HERV-H in a cross-section of the Singapore population and explored the relationship between HERV-H positivity and incidence rates for Hodgkin's lymphoma in three major ethnic groups of Singapore. We observed that Malays were 1.11 times likely (95% CI=1.05–1.17; P<0.01), and Indians 1.12 times likely (95% CI=1.07–1.18; P<0.01) to be HERV-H positive when compared to Chinese. Interestingly, the incidence rates of Hodgkin's lymphoma for the three races positively correlated to the respective prevalence rate for HERV-H positivity (r=0.9921 for male; r=0.9801 for female), suggesting that viral inheritance in human may predispose certain racial origin unfavourably to malignancy

Simplified large-scale Sanger genome sequencing for influenza A/H3N2 virus.

BACKGROUND: The advent of next-generation sequencing technologies and the resultant lower costs of sequencing have enabled production of massive amounts of data, including the generation of full genome sequences of pathogens. However, the small genome size of the influenza virus arguably justifies the use of the more conventional Sanger sequencing technology which is still currently more readily available in most diagnostic laboratories. RESULTS: We present a simplified Sanger-based genome sequencing method for sequencing the influenza A/H3N2 virus in a large-scale format. The entire genome sequencing was completed with 19 reverse transcription-polymerase chain reactions (RT-PCRs) and 39 sequencing reactions. This method was tested on 15 native clinical samples and 15 culture isolates, respectively, collected between 2009 and 2011. The 15 native clinical samples registered quantification cycle values ranging from 21.0 to 30.56, which were equivalent to 2.4×10(3)-1.4×10(6) viral copies/µL of RNA extract. All the PCR-amplified products were sequenced directly without PCR product purification. Notably, high quality sequencing data up to 700 bp were generated for all the samples tested. The completed sequence covered 408,810 nucleotides in total, with 13,627 nucleotides per genome, attaining 100% coding completeness. Of all the bases produced, an average of 89.49% were Phred quality value 40 (QV40) bases (representing an accuracy of circa one miscall for every 10,000 bases) or higher, and an average of 93.46% were QV30 bases (one miscall every 1000 bases) or higher. CONCLUSIONS: This sequencing protocol has been shown to be cost-effective and less labor-intensive in obtaining full influenza genomes. The constant high quality of sequences generated imparts confidence in extending the application of this non-purified amplicon sequencing approach to other gene sequencing assays, with appropriate use of suitably designed primers

PCR primers and second annealing temperatures (T_aS) used to amplify the influenza A/H3N2 genome.

<p>The T_aS for all the PCR primers ranged between 58 and 61°C. MBTuni-12 and MBTuni-13 primers targeting the 5′ and 3′ ends of each segment were adopted from published methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064785#pone.0064785-Chan1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064785#pone.0064785-Zhou1" target="_blank">[27]</a>, with nucleotides (in bold) representing the modifications made. Nucleotide R (bold) in the primer sequence indicates a degenerate nucleotide that represents A or G.</p

Summary of sequencing primers employed in this study and their respective performance.

<p>The performance of each sequencing primer is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064785#pone-0064785-t001" target="_blank">Table 1</a>, as seen by the average percentage of bases generated from the 30 complete genomes with QV more than 30 and 40, respectively. The QV values were generated using the proprietary sequencing analysis software (version 5.2) of the ABI 3130×l genetic analyzer (Applied Biosystems). Length of Read (LOR) is defined as the length of sequence with QV20 and above for at least 20 continuous bases.</p

Molecular epidemiology of rhinovirus among hospitalised patients, Singapore

Human rhinovirus (HRV) is the most prevalent respiratory etiological agent in the world. Over 100 genotypes have been characterised using molecular genotyping techniques. Here, we characterised the molecular epidemiology of the circulating rhinoviruses among hospitalised patients in Singapore by sequencing 134 rhinovirus-positive respiratory specimens that were collected in the period between 2013 and 2015. Each sequence was assigned a genogroup and a genotype using the Enterovirus Genotyping Tool Version 0.1 and phylogenetic reconstruction, respectively. In this study, HRV-A (n=88) and HRV-C (n=38) were identified as the dominant genogroups in Singapore. HRV-A28 (n=7) was the dominant genotype in HRV-A while both HRVC2 (n=8) and HRV-C11 (n=8) were the dominant genotypes in HRV-C. HRV-B was observed to have the lowest number of positive detections in our study population (n=8). The result is interesting as another group had previously found HRV-B to be the second most common genogroup in Singapore after HRV-A

NONINVASIVE RENAL TRANSPLANT GRAFT MONITORING IN A SINGLE INSTITUTION USING CELL-FREE DONOR NUCLEIC ACID IN RECIPIENT PLASMA VIA INSERTION-DELETION ALLELE POLYMORPHISM

10.1016/j.juro.2016.02.1305Annual Meeting of the American-Urological-Association (AUA)1954E431-E43