NanoAmpli-Seq v1.0 : A de novo protocol for amplicon sequencing from mixed microbial communities on the nanopore sequencing platform.
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PROVIDER: PRJEB21005 | ENA |
REPOSITORIES: ENA
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Project description:Amplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences sequencing platforms overcome this limitation, their application has been limited due to higher error rates or lower data output. In this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on the intramolecular-ligated nanopore consensus sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the INC-Seq protocol that reduces sample processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain accurate full-length 16S rRNA gene sequences. NanoAmpli-Seq accurately estimates the diversity of tested mock communities with average consensus sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware base calling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.
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Project description:Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific "conserved" primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.
| S-EPMC3984086 | biostudies-literature
Project description:Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples' genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.
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Project description:We used deep sequencing of the 16S rRNA gene from sputum to identify Haemophilus influenza in a patient with community-acquired pneumonia. This method may be more effective than conventional diagnostic tests in pneumonia patients because of its speed and sensitivity.
| S-EPMC6154133 | biostudies-literature
Project description:ObjectiveWe aimed to evaluate the performance of nanopore amplicon sequencing detection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples.MethodWe carried out a single-center, prospective cohort study in a Wuhan hospital and collected a total of 86 clinical samples, including 54 pharyngeal swabs, 31 sputum samples, and 1 fecal sample, from 86 patients with coronavirus disease 2019 (COVID-19) from Feb 20 to May 15, 2020. We performed parallel detection with nanopore-based genome amplification and sequencing (NAS) on the Oxford Nanopore Technologies (ONT) minION platform and routine reverse transcription quantitative polymerase chain reaction (RT-qPCR). In addition, 27 negative control samples were detected using the two methods. The sensitivity and specificity of NAS were evaluated and compared with those of RT-qPCR.ResultsThe viral read number and reference genome coverage were both significantly different between the two groups of samples, and the latter was a better indicator for SARS-CoV-2 detection. Based on the reference genome coverage, NAS revealed both high sensitivity (96.5%) and specificity (100%) compared with RT-qPCR (80.2 and 96.3%, respectively), although the samples had been stored for half a year before the detection. The total time cost was less than 15 h, which was acceptable compared with that of RT-qPCR (∼2.5 h). In addition, the reference genome coverage of the viral reads was in line with the cycle threshold value of RT-qPCR, indicating that this number could also be used as an indicator of the viral load in a sample. The viral load in sputum might be related to the severity of the infection, particularly in patients within 4 weeks after onset of clinical manifestations, which could be used to evaluate the infection.ConclusionOur results showed the high sensitivity and specificity of the NAS method for SARS-CoV-2 detection compared with RT-qPCR. The sequencing results were also used as an indicator of the viral load to display the viral dynamics during infection. This study proved the wide application prospect of nanopore sequencing detection for SARS-CoV-2 and may more knowledge about the clinical characteristics of COVID-19.
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Project description:A Viral Metagenomic Protocol for Nanopore Sequencing of Group A Rotavirus
| PRJEB53584 | ENA
Project description:Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in COVID-19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS-CoV-2 followed by either massively parallel or nanopore sequencing is a widely-used strategy to obtain genome sequences from primary samples. However, multiplex PCR-based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two-pool, long-amplicon 36-plex PCR primer panel with ~1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2. We validated the panel by assessing nasopharyngeal swab samples with a <30 quantitative reverse transcription PCR cycle threshold value and found that ≥90% of viral genomes could be covered with high sequencing depths (≥20% mean depth). In comparison, the widely-used ARTIC panel yielded 79%-88% high-depth genome regions. We estimated that ~5 Mbp nanopore sequencing data may ensure a >95% viral genome coverage with a ≥10-fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false-positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5' or 3' ends of reads. Thus, nanopore sequencing could not elucidate intra-host viral diversity.
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Project description:DNA adenine methylation identification (DamID) is a powerful method to determine DNA binding profiles of proteins at a genomic scale. The method leverages the fusion between a protein of interest and the Dam methyltransferase of E. coli, which methylates proximal DNA in vivo. Here, we present an optimized procedure, which was developed for tissue-specific analyses in Caenorhabditis elegans and successfully used to footprint genes actively transcribed by RNA polymerases and to map transcription factor binding in gene regulatory regions. The present protocol details C. elegans-specific steps involved in the preparation of transgenic lines and genomic DNA samples, as well as broadly applicable steps for the DamID procedure, including the isolation of methylated DNA fragments, the preparation of multiplexed libraries, Nanopore sequencing, and data analysis. Two distinctive features of the approach are (i) the use of an efficient recombination-based strategy to selectively analyze rare cell types and (ii) the use of Nanopore sequencing, which streamlines the process. The method allows researchers to go from genomic DNA samples to sequencing results in less than a week, while being sensitive enough to report reliable DNA footprints in cell types as rare as 2 cells per animal.
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