scholarly journals Computational Methods for Chromosome-Scale Haplotype Reconstruction

2021 ◽  
Author(s):  
Shilpa Garg
Keyword(s):  
Genetic Variation ◽  
Whole Genome ◽  
Haplotype Reconstruction ◽  
High Quality ◽  
Short Read ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Evolutionary Studies ◽  
Long Read ◽  
Haplotype Information

High-quality chromosome-scale haplotype sequences— of diploid genomes, polyploid genomes and metagenomes — provide important insights into genetic variation associated with disease and biodiversity. However, whole-genome short read sequencing does not yield haplotype information that spans whole chromosomes directly. Computational assembly of shorter haplotype fragments is required for haplotype reconstruction, which can be challenging owing to limited fragment lengths and high haplotype and repeat variability across genomes. Recent advancements in long-read and chromosome-scale sequencing technologies, alongside computational innovations, are improving the reconstruction of haplotypes at the level of whole chromosomes. Here, we review recent methodological progress in these areas and discuss perspectives that could enable routine high-quality haplotype reconstruction in clinical and evolutionary studies.

scholarly journals Computational methods for chromosome-scale haplotype reconstruction

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Shilpa Garg
Keyword(s):  
Genetic Variation ◽  
Computational Methods ◽  
Whole Genome ◽  
Haplotype Reconstruction ◽  
High Quality ◽  
Short Read ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Long Read ◽  
Haplotype Information

AbstractHigh-quality chromosome-scale haplotype sequences of diploid genomes, polyploid genomes, and metagenomes provide important insights into genetic variation associated with disease and biodiversity. However, whole-genome short read sequencing does not yield haplotype information spanning whole chromosomes directly. Computational assembly of shorter haplotype fragments is required for haplotype reconstruction, which can be challenging owing to limited fragment lengths and high haplotype and repeat variability across genomes. Recent advancements in long-read and chromosome-scale sequencing technologies, alongside computational innovations, are improving the reconstruction of haplotypes at the level of whole chromosomes. Here, we review recent and discuss methodological progress and perspectives in these areas.

scholarly journals Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease

2018 ◽  
Author(s):  
Mark T. W. Ebbert ◽  
Stefan Farrugia ◽  
Jonathon Sens ◽  
Karen Jansen-West ◽  
Tania F. Gendron ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Repeat Expansion ◽  
Whole Genome ◽  
Short Read ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Long Read ◽  
Repeat Expansions ◽  
Targeted Approach

AbstractBackground: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.Results: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinlON was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8x coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained >800x coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual’s repeat region was >99% G4C2 content, though we cannot rule out small interruptions.Conclusions: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

scholarly journals Comprehensive identification of transposable element insertions using multiple sequencing technologies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chong Chu ◽  
Rebeca Borges-Monroy ◽  
Vinayak V. Viswanadham ◽  
Soohyun Lee ◽  
Heng Li ◽  
...  
Keyword(s):  
Transposable Element ◽  
Structure And Function ◽  
Endogenous Retroviruses ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Sequencing Data ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read ◽  
And Function

AbstractTransposable elements (TEs) help shape the structure and function of the human genome. When inserted into some locations, TEs may disrupt gene regulation and cause diseases. Here, we present xTea (x-Transposable element analyzer), a tool for identifying TE insertions in whole-genome sequencing data. Whereas existing methods are mostly designed for short-read data, xTea can be applied to both short-read and long-read data. Our analysis shows that xTea outperforms other short read-based methods for both germline and somatic TE insertion discovery. With long-read data, we created a catalogue of polymorphic insertions with full assembly and annotation of insertional sequences for various types of retroelements, including pseudogenes and endogenous retroviruses. Notably, we find that individual genomes have an average of nine groups of full-length L1s in centromeres, suggesting that centromeres and other highly repetitive regions such as telomeres are a significant yet unexplored source of active L1s. xTea is available at https://github.com/parklab/xTea.

scholarly journals Systematic analysis of dark and camouflaged genes: disease-relevant genes hiding in plain sight

2019 ◽  
Author(s):  
Mark T. W. Ebbert ◽  
Tanner D. Jensen ◽  
Karen Jansen-West ◽  
Jonathon P. Sens ◽  
Joseph S. Reddy ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Sequencing Data ◽  
Systematic Analysis ◽  
Protein Coding ◽  
Short Read ◽  
Sequencing Project ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Long Read

AbstractBackgroundThe human genome contains ‘dark’ gene regions that cannot be adequately assembled or aligned using standard short-read sequencing technologies, preventing researchers from identifying mutations within these gene regions that may be relevant to human disease. Here, we identify regions that are ‘dark by depth’ (few mappable reads) and others that are ‘camouflaged’ (ambiguous alignment), and we assess how well long-read technologies resolve these regions. We further present an algorithm to resolve most camouflaged regions (including in short-read data) and apply it to the Alzheimer’s Disease Sequencing Project (ADSP; 13142 samples), as a proof of principle.ResultsBased on standard whole-genome lllumina sequencing data, we identified 37873 dark regions in 5857 gene bodies (3635 protein-coding) from pathways important to human health, development, and reproduction. Of the 5857 gene bodies, 494 (8.4%) were 100% dark (142 protein-coding) and 2046 (34.9%) were ≥5% dark (628 protein-coding). Exactly 2757 dark regions were in protein-coding exons (CDS) across 744 genes. Long-read sequencing technologies from 10x Genomics, PacBio, and Oxford Nanopore Technologies reduced dark CDS regions to approximately 45.1%, 33.3%, and 18.2% respectively. Applying our algorithm to the ADSP, we rescued 4622 exonic variants from 501 camouflaged genes, including a rare, ten-nucleotide frameshift deletion in CR1, a top Alzheimer’s disease gene, found in only five ADSP cases and zero controls.ConclusionsWhile we could not formally assess the CR1 frameshift mutation in Alzheimer’s disease (insufficient sample-size), we believe it merits investigating in a larger cohort. There remain thousands of potentially important genomic regions overlooked by short-read sequencing that are largely resolved by long-read technologies.

scholarly journals Impact of short-read sequencing on the misassembly of a plant genome

2021 ◽  
Author(s):  
Peipei Wang ◽  
Fanrui Meng ◽  
Bethany M. Moore ◽  
Shin-Han Shiu
Keyword(s):  
Great Majority ◽  
Plant Genome ◽  
High Coverage ◽  
Short Read ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Long Read ◽  
Downstream Analysis ◽  
Genomic Regions ◽  
Simple Sequence

Abstract Background: Availability of plant genome sequences has led to significant advances. However, with few exceptions, the great majority of existing genome assemblies are derived from short read sequencing technologies with highly uneven read coverages indicative of sequencing and assembly issues that could significantly impact any downstream analysis of plant genomes. In tomato for example, 0.6% (5.1 Mb) and 9.7% (79.6 Mb) of short-read based assembly had significantly higher and lower coverage compared to background, respectively.Results: To understand what the causes may be for such uneven coverage, we first established machine learning models capable of predicting genomic regions with variable coverages and found that high coverage regions tend to have higher simple sequence repeat and tandem gene densities compared to background regions. To determine if the high coverage regions were misassembled, we examined a recently available tomato long-read based assembly and found that 27.8% (1.41 Mb) of high coverage regions were potentially misassembled of duplicate sequences, compared to 1.4% in background regions. In addition, using a predictive model that can distinguish correctly and incorrectly assembled high coverage regions, we found that misassembled, high coverage regions tend to be flanked by simple sequence repeats, pseudogenes, and transposon elements. Conclusions: Our study provides insights on the causes of variable coverage regions and a quantitative assessment of factors contributing to plant genome misassembly when using short reads.

scholarly journals NextSV: a meta-caller for structural variants from low-coverage long-read sequencing data

2016 ◽  
Author(s):  
Li Fang ◽  
Jiang Hu ◽  
Depeng Wang ◽  
Kai Wang
Keyword(s):  
Whole Genome ◽  
Ashkenazi Jewish ◽  
Structural Variants ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Human Genomes ◽  
Long Read ◽  
Personal Genomes ◽  
Low Coverage

AbstractBackgroundStructural variants (SVs) in human genomes are implicated in a variety of human diseases. Long-read sequencing delivers much longer read lengths than short-read sequencing and may greatly improve SV detection. However, due to the relatively high cost of long-read sequencing, it is unclear what coverage is needed and how to optimally use the aligners and SV callers.ResultsIn this study, we developed NextSV, a meta-caller to perform SV calling from low coverage long-read sequencing data. NextSV integrates three aligners and three SV callers and generates two integrated call sets (sensitive/stringent) for different analysis purposes. We evaluated SV calling performance of NextSV under different PacBio coverages on two personal genomes, NA12878 and HX1. Our results showed that, compared with running any single SV caller, NextSV stringent call set had higher precision and balanced accuracy (F1 score) while NextSV sensitive call set had a higher recall. At 10X coverage, the recall of NextSV sensitive call set was 93.5% to 94.1% for deletions and 87.9% to 93.2% for insertions, indicating that ~10X coverage might be an optimal coverage to use in practice, considering the balance between the sequencing costs and the recall rates. We further evaluated the Mendelian errors on an Ashkenazi Jewish trio dataset.ConclusionsOur results provide useful guidelines for SV detection from low coverage whole-genome PacBio data and we expect that NextSV will facilitate the analysis of SVs on long-read sequencing data.

scholarly journals Impact of short-read sequencing on the misassembly of a plant genome

2020 ◽  
Author(s):  
Peipei Wang ◽  
Fanrui Meng ◽  
Bethany M. Moore ◽  
Shin-Han Shiu
Keyword(s):  
Great Majority ◽  
Plant Genome ◽  
High Coverage ◽  
Short Read ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Long Read ◽  
Downstream Analysis ◽  
Genomic Regions ◽  
Simple Sequence

Abstract Background Availability of plant genome sequences has led to significant advances. However, with few exceptions, the great majority of existing genome assemblies are derived from short read sequencing technologies with highly uneven read coverages indicative of sequencing and assembly issues that could significantly impact any downstream analysis of plant genomes. In tomato for example, 0.6% (5.1 Mb) and 9.7% (79.6 Mb) of short-read based assembly had significantly higher and lower coverage compared to background, respectively. Results To understand what the causes may be for such uneven coverage, we first established machine learning models capable of predicting genomic regions with variable coverages and found that high coverage regions tend to have higher simple sequence repeat and tandem gene densities compared to background regions. To determine if the high coverage regions were misassembled, we examined a recently available tomato long-read based assembly and found that 27.8% (1.41 Mb) of high coverage regions were potentially misassembled of duplicate sequences, compared to 1.4% in background regions. In addition, using a predictive model that can distinguish correctly and incorrectly assembled high coverage regions, we found that misassembled, high coverage regions tend to be flanked by simple sequence repeats, pseudogenes, and transposon elements. Conclusions Our study provides insights on the causes of variable coverage regions and a quantitative assessment of factors contributing to plant genome misassembly when using short reads.

scholarly journals Genomic evaluation of Bordetella spp. originating from Australia

2021 ◽  
Author(s):  
Winkie Fong ◽  
Verlaine Timms ◽  
Eby Sim ◽  
Vitali Sintchenko
Keyword(s):  
Public Health ◽  
Genomic Structure ◽  
Vaccine Uptake ◽  
Snp Analysis ◽  
Whole Genome ◽  
Molecular Monitoring ◽  
Insertion Element ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Read

AbstractBordetella pertussis is the primary causative agent of pertussis, a highly infectious respiratory disease associated with prolonged coughing episodes. Pertussis infections are typically mild in adults, however in neonates, infections can be fatal. Despite successful vaccine uptake, the disease is re-emerging across the globe, therefore it is critical to determine the mechanism by which B. pertussis is escaping vaccination control. Studies have suggested that significant changes have occurred in B. pertussis genomes in response to whole cell and acellular vaccines. Continued molecular monitoring is therefore crucial for public health surveillance.High-resolution molecular surveillance of B. pertussis can be achieved through the sequencing of the whole genome. In public health laboratories, whole genome sequencing is primarily performed by short-read sequencing technologies as they are most cost-effective. However short read sequencing does not resolve the extensive genomic rearrangement evident in Bordetella genomes. This is because repeat regions present in Bordetella genomes are collapsed by downstream analysis. For example, the B. pertussis genome contains more than 200 copies of the IS481 insertion element, hence assemblies generally consist of >200 contigs. Advancements in long-read technologies however increase the potential to circularise and close genomes by bridging the locations of the IS481 insertion element.In this study, we aimed to contextualise the Bordetella spp. circulating in NSW, Australia and assess their relationship with global isolates utilising core genome, SNP and structural clustering analysis using long read technology. We report five closed genomes of Bordetella spp. isolated from Australian patients. Two of the three B. pertussis closed isolates, were unique with their own genomic structure, while the other structurally clustered with global isolates. We found that Australian B. holmesii and B. parapertussis strains cluster with global isolates and do not appear to be unique to Australia. Australian draft B. holmesii SNP analysis showed that between 1999 and 2007, isolates were relatively similar, however post-2012, isolates were distinct from each other. The closed isolates can also be used as high-quality reference sequences for both surveillance and other investigations into pertussis spread.

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

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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