scholarly journals Reference-free reconstruction and quantification of transcriptomes from Nanopore long-read sequencing

2020 ◽  
Author(s):  
Ivan de la Rubia ◽  
Joel A. Indi ◽  
Silvia Carbonell-Sala ◽  
Julien Lagarde ◽  
M Mar Albà ◽  
...  
Keyword(s):  
Single Molecule ◽  
Reference Genome ◽  
Simulated Data ◽  
Cost Effective ◽  
Dna Assembly ◽  
Sequencing Data ◽  
Consensus Sequences ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read

AbstractSingle-molecule long-read sequencing with Nanopore provides an unprecedented opportunity to measure transcriptomes from any sample1–3. However, current analysis methods rely on the comparison with a reference genome or transcriptome2,4,5, or the use of multiple sequencing technologies6,7, thereby precluding cost-effective studies in species with no genome assembly available, in individuals underrepresented in the existing reference, and for the discovery of disease-specific transcripts not directly identifiable from a reference genome. Methods for DNA assembly8–10 cannot be directly transferred to transcriptomes since their consensus sequences lack the required interpretability for genes with multiple transcript isoforms. To address these challenges, we have developed RATTLE, the first tool to perform reference-free reconstruction and quantification of transcripts from Nanopore long reads. Using simulated data, isoform spike-ins, and sequencing data from tissues and cell lines, we demonstrate that RATTLE accurately determines transcript sequence and abundance, is comparable to reference-based methods, and shows saturation in the number of predicted transcripts with increasing number of input reads.

scholarly journals SVIM: Structural Variant Identification using Mapped Long Reads

2018 ◽  
Author(s):  
David Heller ◽  
Martin Vingron
Keyword(s):  
Single Molecule ◽  
Simulated Data ◽  
Structural Variants ◽  
Human Phenotype ◽  
Structural Variant ◽  
Small Indels ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read

AbstractMotivationStructural variants are defined as genomic variants larger than 50bp. They have been shown to affect more bases in any given genome than SNPs or small indels. Additionally, they have great impact on human phenotype and diversity and have been linked to numerous diseases. Due to their size and association with repeats, they are difficult to detect by shotgun sequencing, especially when based on short reads. Long read, single molecule sequencing technologies like those offered by Pacific Biosciences or Oxford Nanopore Technologies produce reads with a length of several thousand base pairs. Despite the higher error rate and sequencing cost, long read sequencing offers many advantages for the detection of structural variants. Yet, available software tools still do not fully exploit the possibilities.ResultsWe present SVIM, a tool for the sensitive detection and precise characterization of structural variants from long read data. SVIM consists of three components for the collection, clustering and combination of structural variant signatures from read alignments. It discriminates five different variant classes including similar types, such as tandem and interspersed duplications and novel element insertions. SVIM is unique in its capability of extracting both the genomic origin and destination of duplications. It compares favorably with existing tools in evaluations on simulated data and real datasets from PacBio and Nanopore sequencing machines.Availability and implementationThe source code and executables of SVIM are available on Github: github.com/eldariont/svim. SVIM has been implemented in Python 3 and published on bioconda and the Python Package [email protected]

scholarly journals Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
High Quality ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Abstract Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

scholarly journals De novo Assembly of the Brugia malayi Genome Using Long Reads from a Single MinION Flowcell

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joseph R. Fauver ◽  
John Martin ◽  
Gary J. Weil ◽  
Makedonka Mitreva ◽  
Peter U. Fischer
Keyword(s):  
Single Molecule ◽  
New Technologies ◽  
Reference Genome ◽  
De Novo ◽  
Complete Mitochondrial Genome ◽  
Nuclear Genome ◽  
Brugia Malayi ◽  
Field Isolates ◽  
Sequencing Technologies ◽  
Long Reads

AbstractFilarial nematode infections cause a substantial global disease burden. Genomic studies of filarial worms can improve our understanding of their biology and epidemiology. However, genomic information from field isolates is limited and available reference genomes are often discontinuous. Single molecule sequencing technologies can reduce the cost of genome sequencing and long reads produced from these devices can improve the contiguity and completeness of genome assemblies. In addition, these new technologies can make generation and analysis of large numbers of field isolates feasible. In this study, we assessed the performance of the Oxford Nanopore Technologies MinION for sequencing and assembling the genome of Brugia malayi, a human parasite widely used in filariasis research. Using data from a single MinION flowcell, a 90.3 Mb nuclear genome was assembled into 202 contigs with an N50 of 2.4 Mb. This assembly covered 96.9% of the well-defined B. malayi reference genome with 99.2% identity. The complete mitochondrial genome was obtained with individual reads and the nearly complete genome of the endosymbiotic bacteria Wolbachia was assembled alongside the nuclear genome. Long-read data from the MinION produced an assembly that approached the quality of a well-established reference genome using comparably fewer resources.

scholarly journals LRSDAY: Long-read Sequencing Data Analysis for Yeasts

2017 ◽  
Author(s):  
Jia-Xing Yue ◽  
Gianni Liti
Keyword(s):  
Genome Assembly ◽  
Model Organism ◽  
Sequencing Data ◽  
Protein Coding ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read ◽  
Downstream Analysis ◽  
Eukaryotic Organisms ◽  
Genomic Regions

AbstractLong-read sequencing technologies have become increasingly popular in genome projects due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast, Saccharomyces cerevisiae, has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here we present LRSDAY, the first one-stop solution to streamline this process. LRSDAY can produce chromosome-level end-to-end genome assembly and comprehensive annotations for various genomic features (including centromeres, protein-coding genes, tRNAs, transposable elements and telomere-associated elements) that are ready for downstream analysis. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable for virtually any eukaryotic organisms. Applying LRSDAY to a S. cerevisiae strain takes ∼43 hrs to generate a complete and well-annotated genome from ∼100X Pacific Biosciences (PacBio) reads using four threads.

scholarly journals Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

2021 ◽  
Vol 12 ◽  
Author(s):  
Davide Bolognini ◽  
Alberto Magi
Keyword(s):  
Variant Calling ◽  
Research Report ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Factors Affecting ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Sequencing Studies ◽  
Long Read

Structural variants (SVs) are genomic rearrangements that involve at least 50 nucleotides and are known to have a serious impact on human health. While prior short-read sequencing technologies have often proved inadequate for a comprehensive assessment of structural variation, more recent long reads from Oxford Nanopore Technologies have already been proven invaluable for the discovery of large SVs and hold the potential to facilitate the resolution of the full SV spectrum. With many long-read sequencing studies to follow, it is crucial to assess factors affecting current SV calling pipelines for nanopore sequencing data. In this brief research report, we evaluate and compare the performances of five long-read SV callers across four long-read aligners using both real and synthetic nanopore datasets. In particular, we focus on the effects of read alignment, sequencing coverage, and variant allele depth on the detection and genotyping of SVs of different types and size ranges and provide insights into precision and recall of SV callsets generated by integrating the various long-read aligners and SV callers. The computational pipeline we propose is publicly available at https://github.com/davidebolo1993/EViNCe and can be adjusted to further evaluate future nanopore sequencing datasets.

scholarly journals Assembly of chromosome-scale contigs by efficiently resolving repetitive sequences with long reads

2018 ◽  
Author(s):  
Huilong Du ◽  
Chengzhi Liang
Keyword(s):  
Single Molecule ◽  
High Efficiency ◽  
Reference Genome ◽  
Repetitive Sequences ◽  
Sequencing Data ◽  
High Quality ◽  
Single Molecule Sequencing ◽  
Genome Maps ◽  
Long Reads ◽  
Novel Method

AbstractDue to the large number of repetitive sequences in complex eukaryotic genomes, fragmented and incompletely assembled genomes lose value as reference sequences, often due to short contigs that cannot be anchored or mispositioned onto chromosomes. Here we report a novel method Highly Efficient Repeat Assembly (HERA), which includes a new concept called a connection graph as well as algorithms for constructing the graph. HERA resolves repeats at high efficiency with single-molecule sequencing data, and enables the assembly of chromosome-scale contigs by further integrating genome maps and Hi-C data. We tested HERA with the genomes of rice R498, maize B73, human HX1 and Tartary buckwheat Pinku1. HERA can correctly assemble most of the tandemly repetitive sequences in rice using single-molecule sequencing data only. Using the same maize and human sequencing data published by Jiao et al. (2017) and Shi et al. (2016), respectively, we dramatically improved on the sequence contiguity compared with the published assemblies, increasing the contig N50 from 1.3 Mb to 61.2 Mb in maize B73 assembly and from 8.3 Mb to 54.4 Mb in human HX1 assembly with HERA. We provided a high-quality maize reference genome with 96.9% of the gaps filled (only 76 gaps left) and several incorrectly positioned sequences fixed compared with the B73 RefGen_v4 assembly. Comparisons between the HERA assembly of HX1 and the human GRCh38 reference genome showed that many gaps in GRCh38 could be filled, and that GRCh38 contained some potential errors that could be fixed. We assembled the Pinku1 genome into 12 scaffolds with a contig N50 size of 27.85 Mb. HERA serves as a new genome assembly/phasing method to generate high quality sequences for complex genomes and as a curation tool to improve the contiguity and completeness of existing reference genomes, including the correction of assembly errors in repetitive regions.

Critical assessment of bioinformatics methods for the characterization of pathological repeat expansions with single-molecule sequencing data

2019 ◽  
Vol 21 (6) ◽  
pp. 1971-1986 ◽  
Author(s):  
Matteo Chiara ◽  
Federico Zambelli ◽  
Ernesto Picardi ◽  
David S Horner ◽  
Graziano Pesole
Keyword(s):  
Single Molecule ◽  
Tandem Repeats ◽  
Simulated Data ◽  
Detailed Comparison ◽  
Sequencing Data ◽  
Single Molecule Sequencing ◽  
Sequencing Technologies ◽  
Repeat Expansions

Abstract A number of studies have reported the successful application of single-molecule sequencing technologies to the determination of the size and sequence of pathological expanded microsatellite repeats over the last 5 years. However, different custom bioinformatics pipelines were employed in each study, preventing meaningful comparisons and somewhat limiting the reproducibility of the results. In this review, we provide a brief summary of state-of-the-art methods for the characterization of expanded repeats alleles, along with a detailed comparison of bioinformatics tools for the determination of repeat length and sequence, using both real and simulated data. Our reanalysis of publicly available human genome sequencing data suggests a modest, but statistically significant, increase of the error rate of single-molecule sequencing technologies at genomic regions containing short tandem repeats. However, we observe that all the methods herein tested, irrespective of the strategy used for the analysis of the data (either based on the alignment or assembly of the reads), show high levels of sensitivity in both the detection of expanded tandem repeats and the estimation of the expansion size, suggesting that approaches based on single-molecule sequencing technologies are highly effective for the detection and quantification of tandem repeat expansions and contractions.

scholarly journals ISOdb: A Comprehensive Database of Full-Length Isoforms Generated by Iso-Seq

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Shang-Qian Xie ◽  
Yue Han ◽  
Xiao-Zhou Chen ◽  
Tai-Yu Cao ◽  
Kai-Kai Ji ◽  
...  
Keyword(s):  
Single Molecule ◽  
Full Length ◽  
Public Access ◽  
Transcript Isoforms ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Depth Analysis ◽  
Gene Level ◽  
Long Read ◽  
Full Length Transcript

The accurate landscape of transcript isoforms plays an important role in the understanding of gene function and gene regulation. However, building complete transcripts is very challenging for short reads generated using next-generation sequencing. Fortunately, isoform sequencing (Iso-Seq) using single-molecule sequencing technologies, such as PacBio SMRT, provides long reads spanning entire transcript isoforms which do not require assembly. Therefore, we have developed ISOdb, a comprehensive resource database for hosting and carrying out an in-depth analysis of Iso-Seq datasets and visualising the full-length transcript isoforms. The current version of ISOdb has collected 93 publicly available Iso-Seq samples from eight species and presents the samples in two levels: (1) sample level, including metainformation, long read distribution, isoform numbers, and alternative splicing (AS) events of each sample; (2) gene level, including the total isoforms, novel isoform number, novel AS number, and isoform visualisation of each gene. In addition, ISOdb provides a user interface in the website for uploading sample information to facilitate the collection and analysis of researchers’ datasets. Currently, ISOdb is the first repository that offers comprehensive resources and convenient public access for hosting, analysing, and visualising Iso-Seq data, which is freely available.

scholarly journals A benchmark of structural variation detection by long reads through a realistic simulated model

2020 ◽  
Author(s):  
Nicolas Dierckxsens ◽  
Tong Li ◽  
Joris R. Vermeesch ◽  
Zhi Xie
Keyword(s):  
Structural Variation ◽  
Rapid Evolution ◽  
Detection Methods ◽  
Sequencing Data ◽  
Simulated Model ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read ◽  
Sequencing Platforms ◽  
The Impact

ABSTRACTDespite the rapid evolution of new sequencing technologies, structural variation detection remains poorly ascertained. The high discrepancy between the results of structural variant analysis programs makes it difficult to assess their performance on real datasets. Accurate simulations of structural variation distributions and sequencing data of the human genome are crucial for the development and benchmarking of new tools. In order to gain a better insight into the detection of structural variation with long sequencing reads, we created a realistic simulated model to thoroughly compare SV detection methods and the impact of the chosen sequencing technology and sequencing depth. To achieve this, we developed Sim-it, a straightforward tool for the simulation of both structural variation and long-read data. These simulations from Sim-it revealed the strengths and weaknesses for current available structural variation callers and long read sequencing platforms. Our findings were also supported by the latest structural variation benchmark set developed by the GIAB Consortium. With these findings, we developed a new method (combiSV) that can combine the results from five different SV callers into a superior call set with increased recall and precision. Both Sim-it and combiSV are open source and can be downloaded at https://github.com/ndierckx/.

scholarly journals SVJedi: Genotyping structural variations with long reads

2019 ◽  
Author(s):  
Lolita Lecompte ◽  
Pierre Peterlongo ◽  
Dominique Lavenier ◽  
Claire Lemaitre
Keyword(s):  
Sequencing Data ◽  
Structural Variations ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Clinical Diagnoses ◽  
Long Read ◽  
Reference Sequences ◽  
The One

AbstractMotivationStudies on structural variants (SV) are expanding rapidly. As a result, and thanks to third generation sequencing technologies, the number of discovered SVs is increasing, especially in the human genome. At the same time, for several applications such as clinical diagnoses, it is important to genotype newly sequenced individuals on well defined and characterized SVs. Whereas several SV genotypers have been developed for short read data, there is a lack of such dedicated tool to assess whether known SVs are present or not in a new long read sequenced sample, such as the one produced by Pacific Biosciences or Oxford Nanopore Technologies.ResultsWe present a novel method to genotype known SVs from long read sequencing data. The method is based on the generation of a set of reference sequences that represent the two alleles of each structural variant. Long reads are aligned to these reference sequences. Alignments are then analyzed and filtered out to keep only informative ones, to quantify and estimate the presence of each SV allele and the allele frequencies. We provide an implementation of the method, SVJedi, to genotype insertions and deletions with long reads. The tool has been applied to both simulated and real human datasets and achieves high genotyping accuracy. We also demonstrate that SV genotyping is considerably improved with SVJedi compared to other approaches, namely SV discovery and short read SV genotyping approaches.Availabilityhttps://github.com/llecompte/[email protected]

Sign in / Sign up

Export Citation Format

Share Document