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

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

10.1101/519025 ◽

2019 ◽

Cited By ~ 27

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.

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Complex Structural Variants Resolved by Short-Read and Long-Read Whole Genome Sequencing in Mendelian Disorders

10.1101/281683 ◽

2018 ◽

Cited By ~ 2

Author(s):

Alba Sanchis-Juan ◽

Jonathan Stephens ◽

Courtney E French ◽

Nicholas Gleadall ◽

Karyn Mégy ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

De Novo ◽

Genomic Variation ◽

Mendelian Disease ◽

Whole Genome ◽

Structural Variants ◽

Short Read ◽

Long Read ◽

Complex Structural

AbstractComplex structural variants (cxSVs) are genomic rearrangements comprising multiple structural variants, typically involving three or more breakpoint junctions. They contribute to human genomic variation and can cause Mendelian disease, however they are not typically considered during genetic testing. Here, we investigate the role of cxSVs in Mendelian disease using short-read whole genome sequencing (WGS) data from 1,324 individuals with neurodevelopmental or retinal disorders from the NIHR BioResource project. We present four cases of individuals with a cxSV affecting Mendelian disease-associated genes. Three of the cxSVs are pathogenic: a de novo duplication-inversion-inversion-deletion affecting ARID1B in an individual with Coffin-Siris syndrome, a deletion-inversion-duplication affecting HNRNPU in an individual with intellectual disability and seizures, and a homozygous deletion-inversion-deletion affecting CEP78 in an individual with cone-rod dystrophy. Additionally, we identified a de novo duplication-inversion-duplication overlapping CDKL5 in an individual with neonatal hypoxic-ischaemic encephalopathy. Long-read sequencing technology used to resolve the breakpoints demonstrated the presence of both a disrupted and an intact copy of CDKL5 on the same allele; therefore, it was classified as a variant of uncertain significance. Analysis of sequence flanking all breakpoint junctions in all the cxSVs revealed both microhomology and longer repetitive sequences, suggesting both replication and homology based processes. Accurate resolution of cxSVs is essential for clinical interpretation, and here we demonstrate that long-read WGS is a powerful technology by which to achieve this. Our results show cxSVs are an important although rare cause of Mendelian disease, and we therefore recommend their consideration during research and clinical investigations.

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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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Biodiversity genomics of small metazoans: high quality de novo genomes from single specimens of field-collected and ethanol-preserved springtails

10.1101/2020.08.10.244541 ◽

2020 ◽

Cited By ~ 2

Author(s):

Clément Schneider ◽

Christian Woehle ◽

Carola Greve ◽

Cyrille A. D’Haese ◽

Magnus Wolf ◽

...

Keyword(s):

Genome Sequencing ◽

De Novo ◽

Genetic Model ◽

Nuclear Genome ◽

High Quality ◽

Sequencing Technologies ◽

Natural Product Research ◽

Genome Wide ◽

Long Read ◽

Hmw Dna

ABSTRACTGenome sequencing of all known eukaryotes on Earth promises unprecedented advances in evolutionary sciences, ecology, systematics and in biodiversity-related applied fields such as environmental management and natural product research. Advances in DNA sequencing technologies make genome sequencing feasible for many non-genetic model species. However, genome sequencing today relies on large quantities of high quality, high molecular weight (HMW) DNA which is mostly obtained from fresh tissues. This is problematic for biodiversity genomics of Metazoa as most species are small and yield minute amounts of DNA. Furthermore, briging living specimens to the lab bench not realistic for the majority of species.Here we overcome those difficulties by sequencing two species of springtails (Collembola) from single specimens preserved in ethanol. We used a newly developed, genome-wide amplification-based protocol to generate PacBio libraries for HiFi long-read sequencing.The assembled genomes were highly continuous. They can be considered complete as we recovered over 95% of BUSCOs. Genome-wide amplification does not seem to bias genome recovery. Presence of almost complete copies of the mitochondrial genome in the nuclear genome were pitfalls for automatic assemblers. The genomes fit well into an existing phylogeny of springtails. A neotype is designated for one of the species, blending genome sequencing and creation of taxonomic references.Our study shows that it is possible to obtain high quality genomes from small, field-preserved sub-millimeter metazoans, thus making their vast diversity accessible to the fields of genomics.

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

10.1101/209122 ◽

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).

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Perspectives and benefits of high-throughput long-read sequencing in microbial ecology

Applied and Environmental Microbiology ◽

10.1128/aem.00626-21 ◽

2021 ◽

Author(s):

Leho Tedersoo ◽

Mads Albertsen ◽

Sten Anslan ◽

Benjamin Callahan

Keyword(s):

Microbial Ecology ◽

High Throughput ◽

Single Molecule ◽

High Throughput Sequencing ◽

Environmental Dna ◽

Nanopore Sequencing ◽

High Quality ◽

Short Read ◽

Sequencing Technologies ◽

Long Read

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities such as rapid molecular diagnostics and direct RNA sequencing, and both PacBio and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

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Scaffolding of long read assemblies using long range contact information

10.1101/083964 ◽

2016 ◽

Cited By ~ 1

Author(s):

Jay Ghurye ◽

Mihai Pop ◽

Sergey Koren ◽

Chen-Shan Chin

Keyword(s):

De Novo ◽

Chromatin Interaction ◽

Interaction Data ◽

Computationally Efficient ◽

Short Read ◽

Contact Information ◽

Genome Wide ◽

Long Read ◽

Genome Assemblies ◽

Chromosome Level

AbstractMotivationLong read technologies have made a revolution in de novo genome assembly by generating contigs of size orders of magnitude more than that of short read assemblies. Although the assembly contiguity has increased, it still does not span a chromosome or an arm of the chromosome, resulting in an unfinished chromosome level assembly. To address this problem, we develop a scalable and computationally efficient scaffolding method that can boost the contiguity of the assembly by a large extent using genome wide chromatin interaction data such as Hi-C. Particularly, we demonstrate an algorithm that uses Hi-C data for longer-range scaffolding of de novo long read genome assemblies.ResultsWe tested our methods on two long read assemblies of different organisms. We compared our method with previously developed method and show that our approach performs better in terms of accuracy of scaffolding.AvailabilityThe software is available for free use and can be downloaded from here: https://github.com/machinegun/[email protected]

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

10.1101/2020.08.10.245308 ◽

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.

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SVIM: structural variant identification using mapped long reads

Bioinformatics ◽

10.1093/bioinformatics/btz041 ◽

2019 ◽

Vol 35 (17) ◽

pp. 2907-2915 ◽

Cited By ~ 32

Author(s):

David Heller ◽

Martin Vingron

Keyword(s):

Single Molecule ◽

Simulated Data ◽

Supplementary Information ◽

Nucleotide Polymorphisms ◽

Structural Variants ◽

Human Phenotype ◽

Structural Variant ◽

Pacific Biosciences ◽

Sequencing Technologies ◽

Long Read

Abstract Motivation Structural variants are defined as genomic variants larger than 50 bp. They have been shown to affect more bases in any given genome than single-nucleotide polymorphisms or small insertions and deletions. 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. Results We 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 Pacific Biosciences and Nanopore sequencing machines. Availability and implementation The 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 Index. Supplementary information Supplementary data are available at Bioinformatics online.

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De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽

10.12688/f1000research.11146.2 ◽

2018 ◽

Vol 6 ◽

pp. 618 ◽

Cited By ~ 2

Author(s):

Michael Liem ◽

Hans J. Jansen ◽

Ron P. Dirks ◽

Christiaan V. Henkel ◽

G. Paul H. van Heusden ◽

...

Keyword(s):

De Novo ◽

Sequence Data ◽

New Technology ◽

Whole Genome ◽

Nanopore Sequencing ◽

Short Read ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Before And After ◽

Long Read

Background: The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

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