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UK circulating strains of human parainfluenza 3: an amplicon based next generation sequencing method and phylogenetic analysis.

ABSTRACT: Background: Human parainfluenza viruses type 3 (HPIV3) are a prominent cause of respiratory infection with a significant impact in both pediatric and transplant patient cohorts. Currently there is a paucity of whole genome sequence data that would allow for detailed epidemiological and phylogenetic analysis of circulating strains in the UK. Although it is known that HPIV3 peaks annually in the UK, to date there are no whole genome sequences of HPIV3 UK strains available. Methods: Clinical strains were obtained from HPIV3 positive respiratory patient samples collected between 2011 and 2015. These were then amplified using an amplicon based method, sequenced on the Illumina platform and assembled using a new robust bioinformatics pipeline. Phylogenetic analysis was carried out in the context of other epidemiological studies and whole genome sequence data currently available with stringent exclusion of significantly culture-adapted strains of HPIV3. Results: In the current paper we have presented twenty full genome sequences of UK circulating strains of HPIV3 and a detailed phylogenetic analysis thereof. We have analysed the variability along the HPIV3 genome and identified a short hypervariable region in the non-coding segment between the M (matrix) and F (fusion) genes. The epidemiological classifications obtained by using this region and whole genome data were then compared and found to be identical. Conclusions: The majority of HPIV3 strains were observed at different geographical locations and with a wide temporal spread, reflecting the global distribution of HPIV3. Consistent with previous data, a particular subcluster or strain was not identified as specific to the UK, suggesting that a number of genetically diverse strains circulate at any one time. A small hypervariable region in the HPIV3 genome was identified and it was shown that, in the absence of full genome data, this region could be used for epidemiological surveillance of HPIV3.

SUBMITTER: Smielewska A

PROVIDER: S-EPMC6281019 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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UK circulating strains of human parainfluenza 3: an amplicon based next generation sequencing method and phylogenetic analysis.

Smielewska Anna A Emmott Edward E Ranellou Kyriaki K Popay Ashley A Goodfellow Ian I Jalal Hamid H

Wellcome open research 20181126

<b>Background:</b> Human parainfluenza viruses type 3 (HPIV3) are a prominent cause of respiratory infection with a significant impact in both pediatric and transplant patient cohorts. Currently there is a paucity of whole genome sequence data that would allow for detailed epidemiological and phylogenetic analysis of circulating strains in the UK. Although it is known that HPIV3 peaks annually in the UK, to date there are no whole genome sequences of HPIV3 UK strains available. <b>Methods:</b> ...[more]

PMID: 30569021

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Project description:COVID-19 is caused by SARS-CoV-2, several virulent variants of which have emerged since 2019. More than 529 million people have been infected, and at least 6 million have died. Our aim was to develop a fast, accurate, low-cost method for detecting and identifying newly emerging variants of concern (VOCs) that could pose a global threat. The 341-bp DNA sequence of a specific region of the SARS-CoV-2's spike protein was amplified by a one-step PCR on RNA samples from 46 patients. The product was sequenced using next-generation sequencing (NGS). DNA sequences from seven genomes, the original Wuhan isolate and six different representative variants obtained from the GISAID website, were used as references. Complete whole-genome sequences from local isolates were also obtained from the GISAID website, and their RNA was used for comparison. We used an amplicon-based NGS method (termed VOC-NGS) for genotyping and successfully identified all 46 samples. Fifteen (32.6%) were like the original isolate. Twenty-seven were VOCs: nine (19.5%) Alpha, eight (19%) Delta, six (14%) Beta, and four (8.7%) Omicron. Two were variants of interest (VOI): one (2%) Kappa and one (2%) Zeta. Two samples were mixtures of two variants, one of Alpha and Beta and one of Alpha and Delta. The Spearman correlation between whole-genome sequencing (WGS) and VOC-NGS was significant (P < 0.001) with perfect agreement (Kappa = 0.916) for 36/38 (94.7%) samples with VOC-NGS detecting all the known VOCs. Genotyping by VOC-NGS enables rapid screening of high-throughput clinical samples that includes the identification of VOCs and mixtures of variants, at lower cost than WGS. IMPORTANCE The manuscript described SARS-Cov-2 genotyping by VOC-NGS, which presents an ideal balance of accuracy, rapidity, and cost for detecting and globally tracking VOCs and some VOI of SARS-CoV-2. A large number of clinical samples can be tested together. Rapid introduction of new mutations at a specific site of the spike protein necessitates efficient strain detection and identification to enable choice of treatment and the application of vaccination, as well as planning public health policy.

Project description:BackgroundNext-generation sequencing (NGS) has enabled efficient high-resolution typing of human leukocyte antigen (HLA) genes with minimal ambiguity. Most commercially available assays amplify individual or subgroup of HLA genes by long-range PCR followed by library preparation and sequencing. The AllType assay simplifies the workflow by amplifying 11 transplant-relevant HLA genes in one PCR reaction. Here, we report the performance of this unique workflow evaluated using 218 genetically diverse samples.MethodsFive whole genes (HLA-A/B/C/DQA1/DPA1) and six near-whole genes (HLA-DRB1/DRB345/DQB1/DPB1; excluding exon 1 and part of intron 1) were amplified in a multiplexed, long-range PCR. Manual library preparation was performed per manufacturer's protocol, followed by template preparation and chip loading on the Ion Chef, and sequencing on the Ion S5 sequencer. Pre-specified rules for quality control and repeat testing were followed; technologists were blinded to the reference results. The concordance between AllType and reference results was determined at 2-field resolution. We also describe the ranges of input DNA and library concentrations, read number per sample and per locus, and key health metrics in relation to typing results.ResultsThe concordance rates were 98.6%, 99.8% and 99.9% at the sample (n = 218), genotype (n = 1688), and allele (n = 3376) levels, respectively. Three genotypes were discordant, all of which shared the same G group typing results with the reference. Most ambiguous genotypes (116 out of 144, 80.6%) were due to the lack of exon 1 and intron 1 coverage for HLA-DRB1/DRB345/DQB1/DPB1 genes. A broad range of input DNA concentrations and library concentrations were tolerated. Per sample read numbers were adequate for accurate genotyping. Per locus read numbers showed some inter-lot variations, and a trend toward improved inter-locus balance was observed with later lots of reagents.ConclusionThe AllType assay on the Ion Chef/Ion S5 platform offers a robust and efficient workflow for clinical HLA typing at the 2-field resolution. The multiplex PCR strategy simplifies the laboratory procedure without compromising the typing accuracy.

Dataset Information

UK circulating strains of human parainfluenza 3: an amplicon based next generation sequencing method and phylogenetic analysis.

Publications

UK circulating strains of human parainfluenza 3: an amplicon based next generation sequencing method and phylogenetic analysis.

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