scholarly journals Highly-accurate long-read sequencing improves variant detection and assembly of a human genome

2019 ◽  
Author(s):  
Aaron M. Wenger ◽  
Paul Peluso ◽  
William J. Rowell ◽  
Pi-Chuan Chang ◽  
Richard J. Hall ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Short Reads ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read ◽  
Variant Detection ◽  
High Quality Genome ◽  
Circular Consensus Sequencing

AbstractThe major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

scholarly journals Robust Benchmark Structural Variant Calls of An Asian Using the State-of-Art Long Fragment Sequencing Technologies

2020 ◽  
Author(s):  
Xiao Du ◽  
Lili Li ◽  
Fan Liang ◽  
Sanyang Liu ◽  
Wenxin Zhang ◽  
...  
Keyword(s):  
B Lymphocyte ◽  
De Novo ◽  
Structural Variants ◽  
High Confidence ◽  
False Negatives ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Circular Consensus Sequencing

AbstractThe importance of structural variants (SVs) on phenotypes and human diseases is now recognized. Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed, few benchmarking procedures are available to confidently assess their performances in biological and clinical research. To facilitate the validation and application of those approaches, our work established an Asian reference material comprising identified benchmark regions and high-confidence SV calls. We established a high-confidence SV callset with 8,938 SVs in an EBV immortalized B lymphocyte line, by integrating four alignment-based SV callers [from 109× PacBio continuous long read (CLR), 22× PacBio circular consensus sequencing (CCS) reads, 104× Oxford Nanopore long reads, and 114× optical mapping platform (Bionano)] and one de novo assembly-based SV caller using CCS reads. A total of 544 randomly selected SVs were validated by PCR and Sanger sequencing, proofing the robustness of our SV calls. Combining trio-binning based haplotype assemblies, we established an SV benchmark for identification of false negatives and false positives by constructing the continuous high confident regions (CHCRs), which cover 1.46Gb and 6,882 SVs supported by at least one diploid haplotype assembly. Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology, disease, and clinical diagnosis.

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

2017 ◽  
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.

scholarly journals Capture of complete ciliate chromosomes in single sequencing reads reveals widespread chromosome isoforms

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Kelsi A. Lindblad ◽  
Jananan S. Pathmanathan ◽  
Sandrine Moreira ◽  
John R. Bracht ◽  
Robert P. Sebra ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Assembly ◽  
Hybrid Approach ◽  
Rapid Expansion ◽  
Genome Sequences ◽  
Short Reads ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Single Genome ◽  
Long Read

Abstract Background Whole-genome shotgun sequencing, which stitches together millions of short sequencing reads into a single genome, ushered in the era of modern genomics and led to a rapid expansion of the number of genome sequences available. Nevertheless, assembly of short reads remains difficult, resulting in fragmented genome sequences. Ultimately, only a sequencing technology capable of capturing complete chromosomes in a single run could resolve all ambiguities. Even “third generation” sequencing technologies produce reads far shorter than most eukaryotic chromosomes. However, the ciliate Oxytricha trifallax has a somatic genome with thousands of chromosomes averaging only 3.2 kbp, making it an ideal candidate for exploring the benefits of sequencing whole chromosomes without assembly. Results We used single-molecule real-time sequencing to capture thousands of complete chromosomes in single reads and to update the published Oxytricha trifallax JRB310 genome assembly. In this version, over 50% of the completed chromosomes with two telomeres derive from single reads. The improved assembly includes over 12,000 new chromosome isoforms, and demonstrates that somatic chromosomes derive from variable rearrangements between somatic segments encoded up to 191,000 base pairs away. However, while long reads reduce the need for assembly, a hybrid approach that supplements long-read sequencing with short reads for error correction produced the most complete and accurate assembly, overall. Conclusions This assembly provides the first example of complete eukaryotic chromosomes captured by single sequencing reads and demonstrates that traditional approaches to genome assembly can mask considerable structural variation.

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 Impact of Chimera-less Long Reads on Metagenomics of Human Gut Viromes Treated With Multiple Displacement Amplification

2020 ◽  
Author(s):  
Yuya Kiguchi ◽  
Suguru Nishijima ◽  
Naveen Kumar ◽  
Masahira Hattori ◽  
Wataru Suda
Keyword(s):  
De Novo ◽  
Multiple Displacement Amplification ◽  
Read Length ◽  
Human Gut ◽  
Average Read Length ◽  
Sequencing Technologies ◽  
Genomic Complexity ◽  
Long Reads ◽  
Long Read ◽  
The Impact

Abstract Background: The ecological and biological features of the indigenous phage community (virome) in the human gut microbiome are poorly understood, possibly due to many fragmented contigs and fewer complete genomes based on conventional short-read metagenomics. Long-read sequencing technologies have attracted attention as an alternative approach to reconstruct long and accurate contigs from microbial communities. However, the impact of long-read metagenomics on human gut virome analysis has not been well evaluated. Results: Here we present chimera-less PacBio long-read metagenomics of multiple displacement amplification (MDA)-treated human gut virome DNA. The method included the development of a novel bioinformatics tool, SACRA (Split Amplified Chimeric Read Algorithm), which efficiently detects and splits numerous chimeric reads in PacBio reads from the MDA-treated virome samples. SACRA treatment of PacBio reads from five samples markedly reduced the average chimera ratio from 72 to 1.5%, generating chimera-less PacBio reads with an average read-length of 1.8 kb. De novo assembly of the chimera-less long reads generated contigs with an average N50 length of 11.1 kb, whereas those of MiSeq short reads from the same samples were 0.7 kb, dramatically improving contig extension. Alignment of both contig sets generated 378 high-quality merged contigs (MCs) composed of the minimum scaffolds of 434 MiSeq and 637 PacBio contigs, respectively, and also identified numerous MiSeq short fragmented contigs ≤500 bp additionally aligned to MCs, which possibly originated from a small fraction of MiSeq chimeric reads. The alignment also revealed that fragmentations of the scaffolded MiSeq contigs were caused primarily by genomic complexity of the community, including local repeats, hypervariable regions, and highly conserved sequences in and between the phage genomes. We identified 142 complete and near-complete phage genomes including 108 novel genomes, varying from 5 to 185 kb in length, the majority of which were predicted to be Microviridae phages including several variants with homologous but distinct genomes, which were fragmented in MiSeq contigs. Conclusions: Long-read metagenomics coupled with SACRA provides an improved method to reconstruct accurate and extended phage genomes from MDA-treated virome samples of the human gut, and potentially from other environmental virome samples.

scholarly journals Long-read genome sequencing for the diagnosis of neurodevelopmental disorders

2020 ◽  
Author(s):  
Susan M. Hiatt ◽  
James M.J. Lawlor ◽  
Lori H. Handley ◽  
Ryne C. Ramaker ◽  
Brianne B. Rogers ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Neurodevelopmental Disorders ◽  
De Novo ◽  
Genomic Analysis ◽  
Data Sets ◽  
Short Read ◽  
Long Read ◽  
Variant Detection ◽  
Circular Consensus Sequencing

AbstractPurposeExome and genome sequencing have proven to be effective tools for the diagnosis of neurodevelopmental disorders (NDDs), but large fractions of NDDs cannot be attributed to currently detectable genetic variation. This is likely, at least in part, a result of the fact that many genetic variants are difficult or impossible to detect through typical short-read sequencing approaches.MethodsHere, we describe a genomic analysis using Pacific Biosciences circular consensus sequencing (CCS) reads, which are both long (>10 kb) and accurate (>99% bp accuracy). We used CCS on six proband-parent trios with NDDs that were unexplained despite extensive testing, including genome sequencing with short reads.ResultsWe identified variants and created de novo assemblies in each trio, with global metrics indicating these data sets are more accurate and comprehensive than those provided by short-read data. In one proband, we identified a likely pathogenic (LP), de novo L1-mediated insertion in CDKL5 that results in duplication of exon 3, leading to a frameshift. In a second proband, we identified multiple large de novo structural variants, including insertion-translocations affecting DGKB and MLLT3, which we show disrupt MLLT3 transcript levels. We consider this extensive structural variation likely pathogenic.ConclusionThe breadth and quality of variant detection, coupled to finding variants of clinical and research interest in two of six probands with unexplained NDDs strongly support the value of long-read genome sequencing for understanding rare disease.

scholarly journals Fast-SG: An alignment-free algorithm for hybrid assembly

2017 ◽  
Author(s):  
Alex Di Genova ◽  
Gonzalo A. Ruz ◽  
Marie-France Sagot ◽  
Alejandro Maass
Keyword(s):  
De Novo ◽  
Reference Level ◽  
Hybrid Assembly ◽  
Short Read ◽  
Sequencing Technologies ◽  
Alignment Free ◽  
Long Reads ◽  
Long Read ◽  
Definition Of ◽  
Large Genomes

ABSTRACTLong read sequencing technologies are the ultimate solution for genome repeats, allowing near reference level reconstructions of large genomes. However, long read de novo assembly pipelines are computationally intense and require a considerable amount of coverage, thereby hindering their broad application to the assembly of large genomes. Alternatively, hybrid assembly methods which combine short and long read sequencing technologies can reduce the time and cost required to produce de novo assemblies of large genomes. In this paper, we propose a new method, called FAST-SG, which uses a new ultra-fast alignment-free algorithm specifically designed for constructing a scaffolding graph using light-weight data structures. FAST-SG can construct the graph from either short or long reads. This allows the reuse of efficient algorithms designed for short read data and permits the definition of novel modular hybrid assembly pipelines. Using comprehensive standard datasets and benchmarks, we show how FAST-SG outperforms the state-of-the-art short read aligners when building the scaffolding graph, and can be used to extract linking information from either raw or error-corrected long reads. We also show how a hybrid assembly approach using FAST-SG with shallow long read coverage (5X) and moderate computational resources can produce long-range and accurate reconstructions of the genomes of Arabidopsis thaliana (Ler-0) and human (NA12878).

scholarly journals Factorial estimating assembly base errors using k-mer abundance difference (KAD) between short reads and genome assembled sequences

2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Cheng He ◽  
Guifang Lin ◽  
Hairong Wei ◽  
Haibao Tang ◽  
Frank F White ◽  
...  
Keyword(s):  
Copy Number ◽  
Error Rates ◽  
Genome Sequences ◽  
Short Reads ◽  
Sequencing Technologies ◽  
Insertion And Deletion ◽  
Novel Approach ◽  
Long Reads ◽  
Long Read ◽  
Genome Assemblies

Abstract Genome sequences provide genomic maps with a single-base resolution for exploring genetic contents. Sequencing technologies, particularly long reads, have revolutionized genome assemblies for producing highly continuous genome sequences. However, current long-read sequencing technologies generate inaccurate reads that contain many errors. Some errors are retained in assembled sequences, which are typically not completely corrected by using either long reads or more accurate short reads. The issue commonly exists, but few tools are dedicated for computing error rates or determining error locations. In this study, we developed a novel approach, referred to as k-mer abundance difference (KAD), to compare the inferred copy number of each k-mer indicated by short reads and the observed copy number in the assembly. Simple KAD metrics enable to classify k-mers into categories that reflect the quality of the assembly. Specifically, the KAD method can be used to identify base errors and estimate the overall error rate. In addition, sequence insertion and deletion as well as sequence redundancy can also be detected. Collectively, KAD is valuable for quality evaluation of genome assemblies and, potentially, provides a diagnostic tool to aid in precise error correction. KAD software has been developed to facilitate public uses.

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