Dataset Information

NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology.

ABSTRACT: HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.

SUBMITTER: Wright IA

PROVIDER: S-EPMC8534097 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Publications

NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology.

Wright Imogen A IA Delaney Kayla E KE Katusiime Mary Grace K MGK Botha Johannes C JC Engelbrecht Susan S Kearney Mary F MF van Zyl Gert U GU

Cells 20210928 10

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the ...[more]

PMID: 34685559

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Project description:The long-read amplicon provides a species-level solution for the community. With the improvement of nanopore flowcells, the accuracy of Oxford Nanopore Technologies (ONT) R10.4.1 has been substantially enhanced, with an average of approximately 99%. To evaluate its effectiveness on amplicons, three types of microbiomes were analyzed by 16S ribosomal RNA (hereinafter referred to as "16S") amplicon sequencing using Novaseq, Pacbio sequel II, and Nanopore PromethION platforms (R9.4.1 and R10.4.1) in the current study. We showed the error rate, recall, precision, and bias index in the mock sample. The error rate of ONT R10.4.1 was greatly reduced, with a better recall in the case of the synthetic community. Meanwhile, in different types of environmental samples, ONT R10.4.1 analysis resulted in a composition similar to Pacbio data. We found that classification tools and databases influence ONT data. Based on these results, we conclude that the ONT R10.4.1 16S amplicon can also be used for application in environmental samples. IMPORTANCE The long-read amplicon supplies the community with a species-level solution. Due to the high error rate of nanopore sequencing early on, it has not been frequently used in 16S studies. Oxford Nanopore Technologies (ONT) introduced the R10.4.1 flowcell with Q20+ reagent to achieve more than 99% accuracy as sequencing technology advanced. However, there has been no published study on the performance of commercial PromethION sequencers with R10.4.1 flowcells on 16S sequencing or on the impact of accuracy improvement on taxonomy (R9.4.1 to R10.4.1) using 16S ONT data. In this study, three types of microbiomes were investigated by 16S ribosomal RNA (rRNA) amplicon sequencing using Novaseq, Pacbio sequel II, and Nanopore PromethION platforms (R9.4.1 and R10.4.1). In the mock sample, we displayed the error rate, recall, precision, and bias index. We observed that the error rate in ONT R10.4.1 is significantly lower, especially when deletions are involved. First and foremost, R10.4.1 and Pacific Bioscience platforms reveal a similar microbiome in environmental samples. This study shows that the R10.4.1 full-length 16S rRNA sequences allow for species identification of environmental microbiota.

Project description:The advent of high-throughput sequencing techniques has recently provided an astonishing insight into the composition and function of the human microbiome. Next-generation sequencing (NGS) has become the gold standard for advanced microbiome analysis; however, 3rd generation real-time sequencing, such as Oxford Nanopore Technologies (ONT), enables rapid sequencing from several kilobases to >2 Mb with high resolution. Despite the wide availability and the enormous potential for clinical and translational applications, ONT is poorly standardized in terms of sampling and storage conditions, DNA extraction, library creation, and bioinformatic classification. Here, we present a comprehensive analysis pipeline with sampling, storage, DNA extraction, library preparation, and bioinformatic evaluation for complex microbiomes sequenced with ONT. Our findings from buccal and rectal swabs and DNA extraction experiments indicate that methods that were approved for NGS microbiome analysis cannot be simply adapted to ONT. We recommend using swabs and DNA extractions protocols with extended washing steps. Both 16S rRNA and metagenomic sequencing achieved reliable and reproducible results. Our benchmarking experiments reveal thresholds for analysis parameters that achieved excellent precision, recall, and area under the precision recall values and is superior to existing classifiers (Kraken2, Kaiju, and MetaMaps). Hence, our workflow provides an experimental and bioinformatic pipeline to perform a highly accurate analysis of complex microbial structures from buccal and rectal swabs. IMPORTANCE Advanced microbiome analysis relies on sequencing of short DNA fragments from microorganisms like bacteria, fungi, and viruses. More recently, long fragment DNA sequencing of 3rd generation sequencing has gained increasing importance and can be rapidly conducted within a few hours due to its potential real-time sequencing. However, the analysis and correct identification of the microbiome relies on a multitude of factors, such as the method of sampling, DNA extraction, sequencing, and bioinformatic analysis. Scientists have used different protocols in the past that do not allow us to compare results across different studies and research fields. Here, we provide a comprehensive workflow from DNA extraction, sequencing, and bioinformatic workflow that allows rapid and accurate analysis of human buccal and rectal swabs with reproducible protocols. This workflow can be readily applied by many scientists from various research fields that aim to use long-fragment microbiome sequencing.

Dataset Information

NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology.

Publications

NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology.

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