Ultra-low input single tube linked-read library method enables short-read NGS systems to generate highly accurate and economical long-range sequencing information for de novo genome assembly and haplotype phasing

AbstractLong-range sequencing information is required for haplotype phasing, de novo assembly and structural variation detection. Current long-read sequencing technologies can provide valuable long-range information but at a high cost with low accuracy and high DNA input requirement. We have developed a single-tube Transposase Enzyme Linked Long-read Sequencing (TELL-Seq™) technology, which enables a low-cost, high-accuracy and high-throughput short-read next generation sequencer to routinely generate over 100 Kb long-range sequencing information with as little as 0.1 ng input material. In a PCR tube, millions of clonally barcoded beads are used to uniquely barcode long DNA molecules in an open bulk reaction without dilution and compartmentation. The barcode linked reads are used to successfully assemble genomes ranging from microbes to human. These linked-reads also generate mega-base-long phased blocks and provide a cost-effective tool for detecting structural variants in a genome, which are important to identify compound heterozygosity in recessive Mendelian diseases and discover genetic drivers and diagnostic biomarkers in cancers.

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Efficient long single molecule sequencing for cost effective and accurate sequencing, haplotyping, and de novo assembly

10.1101/324392 ◽

2018 ◽

Author(s):

Ou Wang ◽

Robert Chin ◽

Xiaofang Cheng ◽

Michelle Ka Wu ◽

Qing Mao ◽

...

Keyword(s):

Single Molecule ◽

Genome Assembly ◽

De Novo ◽

Low Cost ◽

Variant Calling ◽

Cost Effective ◽

High Quality ◽

Single Molecule Sequencing ◽

Single Tube ◽

Complex Structural

Obtaining accurate sequences from long DNA molecules is very important for genome assembly and other applications. Here we describe single tube long fragment read (stLFR), a technology that enables this a low cost. It is based on adding the same barcode sequence to sub-fragments of the original long DNA molecule (DNA co-barcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process up to 3.6 billion unique barcode sequences were generated on beads, enabling practically non-redundant co-barcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique co-barcoding of over 8 million 20-300 kb genomic DNA fragments. Analysis of the genome of the human genome NA12878 with stLFR demonstrated high quality variant calling and phasing into contigs up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries and their construction did not significantly add to the time or cost of whole genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

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W2RAP: a pipeline for high quality, robust assemblies of large complex genomes from short read data

10.1101/110999 ◽

2017 ◽

Cited By ~ 9

Author(s):

Bernardo J. Clavijo ◽

Gonzalo Garcia Accinelli ◽

Jonathan Wright ◽

Darren Heavens ◽

Katie Barr ◽

...

Keyword(s):

De Novo ◽

Low Cost ◽

Cost Effective ◽

Data Generation ◽

Sequencing Data ◽

High Quality ◽

Crop Species ◽

Short Read ◽

Link Type ◽

Sequencing Technologies

AbstractProducing high-quality whole-genome shotgun de novo assemblies from plant and animal species with large and complex genomes using low-cost short read sequencing technologies remains a challenge. But when the right sequencing data, with appropriate quality control, is assembled using approaches focused on robustness of the process rather than maximization of a single metric such as the usual contiguity estimators, good quality assemblies with informative value for comparative analyses can be produced. Here we present a complete method described from data generation and qc all the way up to scaffold of complex genomes using Illumina short reads and its application to data from plants and human datasets. We show how to use the w2rap pipeline following a metric-guided approach to produce cost-effective assemblies. The assemblies are highly accurate, provide good coverage of the genome and show good short range contiguity. Our pipeline has already enabled the rapid, cost-effective generation of de novo genome assemblies from large, polyploid crop species with a focus on comparative genomics.Availabilityw2rap is available under MIT license, with some subcomponents under GPL-licenses. A ready-to-run docker with all software pre-requisites and example data is also available.http://github.com/bioinfologics/w2raphttp://github.com/bioinfologics/w2rap-contigger

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ReorientExpress: reference-free orientation of nanopore cDNA reads with deep learning

10.1101/553321 ◽

2019 ◽

Author(s):

Angel Ruiz-Reche ◽

Joel A. Indi ◽

Ivan de la Rubia ◽

Eduardo Eyras

Keyword(s):

De Novo ◽

Functional Characterization ◽

Cost Effective ◽

Error Rates ◽

Cdna Libraries ◽

Rna Molecules ◽

Sequencing Technologies ◽

Long Reads ◽

A Genome ◽

Long Read

Long-read sequencing technologies allow the systematic interrogation of transcriptomes from any species. However, functional characterization requires the determination of the correct 5’-to-3’ orientation of reads. Oxford Nanopore Technologies (ONT) allows the direct measurement of RNA molecules in the native orientation (Garalde et al. 2018), but sequencing of complementary-DNA (cDNA) libraries yields generally a larger number of reads (Workman et al. 2018). Although strand-specific adapters can be used, error rates hinder their detection. Current methods rely on the comparison to a genome or transcriptome reference (Wyman and Mortazavi 2018; Workman et al. 2018) or on the use of additional technologies (Fu et al. 2018), which limits the applicability of rapid and cost-effective long-read sequencing for transcriptomics beyond model species. To facilitate the interrogation of transcriptomes de-novo in species or samples for which a genome or transcriptome reference is not available, we have developed ReorientExpress (https://github.com/comprna/reorientexpress), a new tool to perform reference-free orientation of ONT reads from a cDNA library, with our without stranded adapters. ReorientExpress uses a deep neural network (DNN) to predict the orientation of cDNA long-reads independently of adapters and without using a reference.

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Long-read viral metagenomics enables capture of abundant and microdiverse viral populations and their niche-defining genomic islands

10.1101/345041 ◽

2018 ◽

Cited By ~ 3

Author(s):

Joanna Warwick-Dugdale ◽

Natalie Solonenko ◽

Karen Moore ◽

Lauren Chittick ◽

Ann C. Gregory ◽

...

Keyword(s):

High Throughput ◽

Low Cost ◽

Cost Effective ◽

Host Community ◽

Genomic Islands ◽

Viral Metagenomics ◽

Viral Gene ◽

Bioinformatics Pipeline ◽

Short Read ◽

Long Read

AbstractMarine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in culturing of important hosts and the lack of a ‘universal’ gene to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution. However, short-read assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Assembly across genomic islands which likely contain niche-defining genes that drive ecological speciation is also challenging. While such populations and features are successfully captured by single-virus genomics and fosmid-based approaches, both techniques require considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative method for improving assembly of viral metagenomics using long read technology. Named ‘VirION’ (Viral, long-read metagenomics via MinION sequencing), our sequencing approach and complementary bioinformatics pipeline (i) increased number and completeness of assembled viral genomes compared to short-read sequencing methods; (ii) captured populations of abundant viruses with high microdiversity missed by short-read methods and (iii) captured more and longer genomic islands than short-read methods. Thus, VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

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Improved de novo Genome Assembly: Linked-Read Sequencing Combined with Optical Mapping Produce a High Quality Mammalian Genome at Relatively Low Cost

10.1101/128348 ◽

2017 ◽

Cited By ~ 10

Author(s):

DW Mohr ◽

A Naguib ◽

NI Weisenfeld ◽

V Kumar ◽

P Shah ◽

...

Keyword(s):

Genome Assembly ◽

De Novo ◽

Low Cost ◽

Cost Effective ◽

Conserved Synteny ◽

High Quality ◽

De Novo Genome Assembly ◽

Optical Maps ◽

Commercial Applications ◽

Hybrid Scaffolds

AbstractCurrent short-read methods have come to dominate genome sequencing because they are cost-effective, rapid, and accurate. However, short reads are most applicable when data can be aligned to a known reference. Two new methods for de novo assembly are linked-reads and restriction-site labeled optical maps. We combined commercial applications of these technologies for genome assembly of an endangered mammal, the Hawaiian Monk seal.We show that the linked-reads produced with 10X Genomics Chromium chemistry and assembled with Supernova v1.1 software produced scaffolds with an N50 of 22.23 Mbp with the longest individual scaffold of 84.06 Mbp. When combined with Bionano Genomics optical maps using Bionano RefAligner, the scaffold N50 increased to 29.65 Mbp for a total of 170 hybrid scaffolds, the longest of which was 84.78 Mbp. These results were 161X and 215X, respectively, improved over DISCOVAR de novo assemblies. The quality of the scaffolds was assessed using conserved synteny analysis of both the DNA sequence and predicted seal proteins relative to the genomes of humans and other species. We found large blocks of conserved synteny suggesting that the hybrid scaffolds were high quality. An inversion in one scaffold complementary to human chromosome 6 was found and confirmed by optical maps.The complementarity of linked-reads and optical maps is likely to make the production of high quality genomes more routine and economical and, by doing so, significantly improve our understanding of comparative genome biology.

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De Novo Genome Assembly of Limpet Bathyacmaea lactea (Gastropoda: Pectinodontidae): The First Reference Genome of a Deep-Sea Gastropod Endemic to Cold Seeps

Genome Biology and Evolution ◽

10.1093/gbe/evaa100 ◽

2020 ◽

Vol 12 (6) ◽

pp. 905-910 ◽

Cited By ~ 2

Author(s):

Ruoyu Liu ◽

Kun Wang ◽

Jun Liu ◽

Wenjie Xu ◽

Yang Zhou ◽

...

Keyword(s):

Deep Sea ◽

Metal Ion ◽

De Novo ◽

Demographic History ◽

Gene Families ◽

Phylogenetic Position ◽

Cold Seeps ◽

Nitrogen And Phosphorus ◽

De Novo Genome Assembly ◽

A Genome

Abstract Cold seeps, characterized by the methane, hydrogen sulfide, and other hydrocarbon chemicals, foster one of the most widespread chemosynthetic ecosystems in deep sea that are densely populated by specialized benthos. However, scarce genomic resources severely limit our knowledge about the origin and adaptation of life in this unique ecosystem. Here, we present a genome of a deep-sea limpet Bathyacmaea lactea, a common species associated with the dominant mussel beds in cold seeps. We yielded 54.6 gigabases (Gb) of Nanopore reads and 77.9-Gb BGI-seq raw reads, respectively. Assembly harvested a 754.3-Mb genome for B. lactea, with 3,720 contigs and a contig N50 of 1.57 Mb, covering 94.3% of metazoan Benchmarking Universal Single-Copy Orthologs. In total, 23,574 protein-coding genes and 463.4 Mb of repetitive elements were identified. We analyzed the phylogenetic position, substitution rate, demographic history, and TE activity of B. lactea. We also identified 80 expanded gene families and 87 rapidly evolving Gene Ontology categories in the B. lactea genome. Many of these genes were associated with heterocyclic compound metabolism, membrane-bounded organelle, metal ion binding, and nitrogen and phosphorus metabolism. The high-quality assembly and in-depth characterization suggest the B. lactea genome will serve as an essential resource for understanding the origin and adaptation of life in the cold seeps.

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Genotyping structural variants in pangenome graphs using the vg toolkit

10.1101/654566 ◽

2019 ◽

Cited By ~ 7

Author(s):

Glenn Hickey ◽

David Heller ◽

Jean Monlong ◽

Jonas A. Sibbesen ◽

Jouni Sirén ◽

...

Keyword(s):

De Novo ◽

State Of The Art ◽

Effective Means ◽

Point Mutations ◽

Structural Variants ◽

Short Read ◽

Yeast Strains ◽

Sequencing Studies ◽

Long Read

AbstractStructural variants (SVs) remain challenging to represent and study relative to point mutations despite their demonstrated importance. We show that variation graphs, as implemented in the vg toolkit, provide an effective means for leveraging SV catalogs for short-read SV genotyping experiments. We benchmarked vg against state-of-the-art SV genotypers using three sequence-resolved SV catalogs generated by recent long-read sequencing studies. In addition, we use assemblies from 12 yeast strains to show that graphs constructed directly from aligned de novo assemblies improve genotyping compared to graphs built from intermediate SV catalogs in the VCF format.

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Accurate long-read de novo assembly evaluation with Inspector

Genome Biology ◽

10.1186/s13059-021-02527-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yu Chen ◽

Yixin Zhang ◽

Amy Y. Wang ◽

Min Gao ◽

Zechen Chong

Keyword(s):

Genome Assembly ◽

De Novo Assembly ◽

In Silico ◽

Large Scale ◽

De Novo ◽

Small Scale ◽

De Novo Genome Assembly ◽

Consensus Sequences ◽

Assembly Evaluation ◽

Long Read

AbstractLong-read de novo genome assembly continues to advance rapidly. However, there is a lack of effective tools to accurately evaluate the assembly results, especially for structural errors. We present Inspector, a reference-free long-read de novo assembly evaluator which faithfully reports types of errors and their precise locations. Notably, Inspector can correct the assembly errors based on consensus sequences derived from raw reads covering erroneous regions. Based on in silico and long-read assembly results from multiple long-read data and assemblers, we demonstrate that in addition to providing generic metrics, Inspector can accurately identify both large-scale and small-scale assembly errors.

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LRSim: a Linked Reads Simulator generating insights for better genome partitioning

10.1101/103549 ◽

2017 ◽

Cited By ~ 1

Author(s):

Ruibang Luo ◽

Fritz J. Sedlazeck ◽

Charlotte A. Darby ◽

Stephen M. Kelly ◽

Michael C. Schatz

Keyword(s):

Genome Assembly ◽

Fragment Length ◽

De Novo ◽

Library Preparation ◽

Haplotype Phasing ◽

Multiple Datasets ◽

Long Read ◽

Fine Control ◽

Informatics Tools ◽

Sequencing Process

AbstractMotivationLinked reads are a form of DNA sequencing commercialized by 10X Genomics that uses highly multiplexed barcoding within microdroplets to tag short reads to progenitor molecules. The linked reads, spanning tens to hundreds of kilobases, offer an alternative to long-read sequencing for de novo assembly, haplotype phasing and other applications. However, there is no available simulator, making it difficult to measure their capability or develop new informatics tools.ResultsOur analysis of 13 real linked read datasets revealed their characteristics of barcodes, molecules and partitions. Based on this, we introduce LRSim that simulates linked reads by emulating the library preparation and sequencing process with fine control of 1) the number of simulated variants; 2) the linked-read characteristics; and 3) the Illumina reads profile. We conclude from the phasing and genome assembly of multiple datasets, recommendations on coverage, fragment length, and partitioning when sequencing human and non-human genome.AvailabilityLRSIM is under MIT license and is freely available at https://github.com/aquaskyline/[email protected]

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Mapping and phasing of structural variation in patient genomes using nanopore sequencing

10.1101/129379 ◽

2017 ◽

Cited By ~ 4

Author(s):

Mircea Cretu Stancu ◽

Markus J. van Roosmalen ◽

Ivo Renkens ◽

Marleen Nieboer ◽

Sjors Middelkamp ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Structural Variants ◽

Human Genetic Disease ◽

Structural Genomic ◽

Short Read ◽

Sequencing Technologies ◽

Genome Wide ◽

Long Read ◽

Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

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