Browse
Submit Data
Databases
API
Help

Dataset Information

36 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

INC-Seq: accurate single molecule reads using nanopore sequencing.

ABSTRACT: BACKGROUND:Nanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionize genomics. However, several applications are limited by relatively high single-read error rates (>10 %), including RNA-seq, haplotype sequencing and 16S sequencing. RESULTS:We developed the Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) as a strategy for obtaining long and accurate nanopore reads, starting with low input DNA. Applying INC-Seq for 16S rRNA-based bacterial profiling generated full-length amplicon sequences with a median accuracy >97 %. CONCLUSIONS:INC-Seq reads enabled accurate species-level classification, identification of species at 0.1 % abundance and robust quantification of relative abundances, providing a cheap and effective approach for pathogen detection and microbiome profiling on the MinION system.

SUBMITTER: Li C

PROVIDER: S-EPMC4970289 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

INC-Seq: accurate single molecule reads using nanopore sequencing.

Li Chenhao C Chng Kern Rei KR Boey Esther Jia Hui EJ Ng Amanda Hui Qi AH Wilm Andreas A Nagarajan Niranjan N

GigaScience 20160802 1

<h4>Background</h4>Nanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionize genomics. However, several applications are limited by relatively high single-read error rates (>10 %), including RNA-seq, haplotype sequencing and 16S sequencing.<h4>Results</h4>We developed the Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) as a strategy for obtaining long and accurate nanopore reads, starting with low input DNA. Applying ...[more]

PMID: 27485345

Similar Datasets

Long-read single-molecule RNA structure sequencing using nanopore.

Project description:RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.

| S-EPMC9723614 | biostudies-literature

Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing.

Project description:Single Molecule, Real-Time (SMRT) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution.

| S-EPMC4787755 | biostudies-literature

Accurate detection of complex structural variations using single-molecule sequencing.

Project description:Structural variations are the greatest source of genetic variation, but they remain poorly understood because of technological limitations. Single-molecule long-read sequencing has the potential to dramatically advance the field, although high error rates are a challenge with existing methods. Addressing this need, we introduce open-source methods for long-read alignment (NGMLR; https://github.com/philres/ngmlr ) and structural variant identification (Sniffles; https://github.com/fritzsedlazeck/Sniffles ) that provide unprecedented sensitivity and precision for variant detection, even in repeat-rich regions and for complex nested events that can have substantial effects on human health. In several long-read datasets, including healthy and cancerous human genomes, we discovered thousands of novel variants and categorized systematic errors in short-read approaches. NGMLR and Sniffles can automatically filter false events and operate on low-coverage data, thereby reducing the high costs that have hindered the application of long reads in clinical and research settings.

| S-EPMC5990442 | biostudies-literature

Direct detection of RNA modifications and structure using single-molecule nanopore sequencing.

Project description:Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA molecules using chemical probing strategies that reveal RNA structure and dynamics. Many methods exist to detect RNA modifications by short-read sequencing; however, limitations on read length inherent to short-read-based methods dissociate modifications from their native context, preventing single-molecule modification analysis. Here, we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications on long RNAs at the single-molecule level. We detect endogenous 2'-O-methyl and base modifications across E. coli and S. cerevisiae ribosomal RNAs as shifts in current signal and dwell times distally through interactions with the helicase motor protein. We further use the 2'-hydroxyl reactive SHAPE reagent acetylimidazole to probe RNA structure at the single-molecule level with readout by direct nanopore sequencing.

| S-EPMC8896822 | biostudies-literature

Nanopore sequencing of single-cell transcriptomes with scCOLOR-seq.

Project description:Here we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.

| S-EPMC8668430 | biostudies-literature

Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach.

Project description:Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family.

| S-EPMC7261825 | biostudies-literature

Optical recognition of converted DNA nucleotides for single-molecule DNA sequencing using nanopore arrays.

Project description:We demonstrate the feasibility of a nanopore based single-molecule DNA sequencing method, which employs multicolor readout. Target DNA is converted according to a binary code, which is recognized by molecular beacons with two types of fluorophores. Solid-state nanopores are then used to sequentially strip off the beacons, leading to a series of detectable photon bursts, at high speed. We show that signals from multiple nanopores can be detected simultaneously, allowing straightforward parallelization to large nanopore arrays.

| S-EPMC2883017 | biostudies-literature

Accurate mitochondrial DNA sequencing using off-target reads provides a single test to identify pathogenic point mutations.

Project description:PurposeMitochondrial disorders are a common cause of inherited metabolic disease and can be due to mutations affecting mitochondrial DNA or nuclear DNA. The current diagnostic approach involves the targeted resequencing of mitochondrial DNA and candidate nuclear genes, usually proceeds step by step, and is time consuming and costly. Recent evidence suggests that variations in mitochondrial DNA sequence can be obtained from whole-exome sequence data, raising the possibility of a comprehensive single diagnostic test to detect pathogenic point mutations.MethodsWe compared the mitochondrial DNA sequence derived from off-target exome reads with conventional mitochondrial DNA Sanger sequencing in 46 subjects.ResultsMitochondrial DNA sequences can be reliably obtained using three different whole-exome sequence capture kits. Coverage correlates with the relative amount of mitochondrial DNA in the original genomic DNA sample, heteroplasmy levels can be determined using variant and total read depths, and-providing there is a minimum read depth of 20-fold-rare sequencing errors occur at a rate similar to that observed with conventional Sanger sequencing.ConclusionThis offers the prospect of using whole-exome sequence in a diagnostic setting to screen not only all protein coding nuclear genes but also all mitochondrial DNA genes for pathogenic mutations. Off-target mitochondrial DNA reads can also be used to assess quality control and maternal ancestry, inform on ethnic origin, and allow genetic disease association studies not previously anticipated with existing whole-exome data sets.

| S-EPMC4272251 | biostudies-literature

NanoSplicer: accurate identification of splice junctions using Oxford Nanopore sequencing.

Project description:MotivationLong-read sequencing methods have considerable advantages for characterizing RNA isoforms. Oxford Nanopore sequencing records changes in electrical current when nucleic acid traverses through a pore. However, basecalling of this raw signal (known as a squiggle) is error prone, making it challenging to accurately identify splice junctions. Existing strategies include utilizing matched short-read data and/or annotated splice junctions to correct nanopore reads but add expense or limit junctions to known (incomplete) annotations. Therefore, a method that could accurately identify splice junctions solely from nanopore data would have numerous advantages.ResultsWe developed 'NanoSplicer' to identify splice junctions using raw nanopore signal (squiggles). For each splice junction, the observed squiggle is compared to candidate squiggles representing potential junctions to identify the correct candidate. Measuring squiggle similarity enables us to compute the probability of each candidate junction and find the most likely one. We tested our method using (i) synthetic mRNAs with known splice junctions and (ii) biological mRNAs from a lung-cancer cell-line. The results from both datasets demonstrate NanoSplicer improves splice junction identification, especially when the basecalling error rate near the splice junction is elevated.Availability and implementationNanoSplicer is available at https://github.com/shimlab/NanoSplicer and archived at https://doi.org/10.5281/zenodo.6403849. Data is available from ENA: ERS7273757 and ERS7273453.Supplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC9344838 | biostudies-literature

Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory.

Project description:BACKGROUND: Recent methods have been developed to perform high-throughput sequencing of DNA by Single Molecule Sequencing (SMS). While Next-Generation sequencing methods may produce reads up to several hundred bases long, SMS sequencing produces reads up to tens of kilobases long. Existing alignment methods are either too inefficient for high-throughput datasets, or not sensitive enough to align SMS reads, which have a higher error rate than Next-Generation sequencing. RESULTS: We describe the method BLASR (Basic Local Alignment with Successive Refinement) for mapping Single Molecule Sequencing (SMS) reads that are thousands of bases long, with divergence between the read and genome dominated by insertion and deletion error. The method is benchmarked using both simulated reads and reads from a bacterial sequencing project. We also present a combinatorial model of sequencing error that motivates why our approach is effective. CONCLUSIONS: The results indicate that it is possible to map SMS reads with high accuracy and speed. Furthermore, the inferences made on the mapability of SMS reads using our combinatorial model of sequencing error are in agreement with the mapping accuracy demonstrated on simulated reads.

| S-EPMC3572422 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data