NanoSatellite: accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION

AbstractTechnological limitations have hindered the large-scale genetic investigation of tandem repeats in disease. We show that long-read sequencing with a single Oxford Nanopore Technologies PromethION flow cell per individual achieves 30× human genome coverage and enables accurate assessment of tandem repeats including the 10,000-bp Alzheimer’s disease-associated ABCA7 VNTR. The Guppy “flip-flop” base caller and tandem-genotypes tandem repeat caller are efficient for large-scale tandem repeat assessment, but base calling and alignment challenges persist. We present NanoSatellite, which analyzes tandem repeats directly on electric current data and improves calling of GC-rich tandem repeats, expanded alleles, and motif interruptions.

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Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing

10.1101/2021.09.27.21263187 ◽

2021 ◽

Author(s):

Igor Stevanovski ◽

Sanjog R. Chintalaphani ◽

Hasindu Gamaarachchi ◽

James M. Ferguson ◽

Sandy S. Pineda ◽

...

Keyword(s):

Tandem Repeat ◽

Tandem Repeats ◽

Fragile X ◽

Genetic Diagnosis ◽

Neuromuscular Diseases ◽

Nanopore Sequencing ◽

Molecular Tests ◽

Genetic Landscape ◽

Long Read ◽

Short Tandem

ABSTRACTShort-tandem repeat (STR) expansions are an important class of pathogenic genetic variants. Over forty neurological and neuromuscular diseases are caused by STR expansions, with 37 different genes implicated to date. Here we describe the use of programmable targeted long-read sequencing with Oxford Nanopore’s ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single, simple assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of expanded and non-expanded STR sites. In doing so, the assay correctly diagnoses all individuals in a cohort of patients (n = 27) with various neurogenetic diseases, including Huntington’s disease, fragile X syndrome and cerebellar ataxia (CANVAS) and others. Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing, and identifies non-canonical STR motif conformations and internal sequence interruptions. Even in our relatively small cohort, we observe a wide diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of STR expansion disorders. Finally, we show how the flexible inclusion of pharmacogenomics (PGx) genes as secondary ReadUntil targets can identify clinically actionable PGx genotypes to further inform patient care, at no extra cost. Our study addresses the need for improved techniques for genetic diagnosis of STR expansion disorders and illustrates the broad utility of programmable long-read sequencing for clinical genomics.One sentence summaryThis study describes the development and validation of a programmable targeted nanopore sequencing assay for parallel genetic diagnosis of all known pathogenic short-tandem repeats (STRs) in a single, simple test.

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Human-specific tandem repeat expansion and differential gene expression during primate evolution

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1912175116 ◽

2019 ◽

Vol 116 (46) ◽

pp. 23243-23253 ◽

Cited By ~ 13

Author(s):

Arvis Sulovari ◽

Ruiyang Li ◽

Peter A. Audano ◽

David Porubsky ◽

Mitchell R. Vollger ◽

...

Keyword(s):

Tandem Repeat ◽

Tandem Repeats ◽

Sequence Data ◽

Variable Number ◽

Specific Expression ◽

Sequence Composition ◽

Transcription Profiles ◽

Long Read ◽

Repeat Expansions ◽

Human Specific

Short tandem repeats (STRs) and variable number tandem repeats (VNTRs) are important sources of natural and disease-causing variation, yet they have been problematic to resolve in reference genomes and genotype with short-read technology. We created a framework to model the evolution and instability of STRs and VNTRs in apes. We phased and assembled 3 ape genomes (chimpanzee, gorilla, and orangutan) using long-read and 10x Genomics linked-read sequence data for 21,442 human tandem repeats discovered in 6 haplotype-resolved assemblies of Yoruban, Chinese, and Puerto Rican origin. We define a set of 1,584 STRs/VNTRs expanded specifically in humans, including large tandem repeats affecting coding and noncoding portions of genes (e.g., MUC3A, CACNA1C). We show that short interspersed nuclear element–VNTR–Alu (SVA) retrotransposition is the main mechanism for distributing GC-rich human-specific tandem repeat expansions throughout the genome but with a bias against genes. In contrast, we observe that VNTRs not originating from retrotransposons have a propensity to cluster near genes, especially in the subtelomere. Using tissue-specific expression from human and chimpanzee brains, we identify genes where transcript isoform usage differs significantly, likely caused by cryptic splicing variation within VNTRs. Using single-cell expression from cerebral organoids, we observe a strong effect for genes associated with transcription profiles analogous to intermediate progenitor cells. Finally, we compare the sequence composition of some of the largest human-specific repeat expansions and identify 52 STRs/VNTRs with at least 40 uninterrupted pure tracts as candidates for genetically unstable regions associated with disease.

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Chasing perfection: validation and polishing strategies for telomere-to-telomere genome assemblies

10.21203/rs.3.rs-712747/v1 ◽

2021 ◽

Author(s):

Arang Rhie ◽

Ann Mc Cartney ◽

Kishwar Shafin ◽

Michael Alonge ◽

Andrey Bzikadze ◽

...

Keyword(s):

Genome Assembly ◽

Tandem Repeats ◽

Hydatidiform Mole ◽

Segmental Duplications ◽

Sequencing Technologies ◽

Oxford Nanopore ◽

Human Genome Assembly ◽

Long Read ◽

Genome Assemblies ◽

Oxford Nanopore Technologies

Abstract Advances in long-read sequencing technologies and genome assembly methods have enabled the recent completion of the first Telomere-to-Telomere (T2T) human genome assembly, which resolves complex segmental duplications and large tandem repeats, including centromeric satellite arrays in a complete hydatidiform mole (CHM13). Though derived from highly accurate sequencing, evaluation revealed that the initial T2T draft assembly had evidence of small errors and structural misassemblies. To correct these errors, we designed a novel repeat-aware polishing strategy that made accurate assembly corrections in large repeats without overcorrection, ultimately fixing 51% of the existing errors and improving the assembly QV to 73.9. By comparing our results to standard automated polishing tools, we outline common polishing errors and offer practical suggestions for genome projects with limited resources. We also show how sequencing biases in both PacBio HiFi and Oxford Nanopore Technologies reads cause signature assembly errors that can be corrected with a diverse panel of sequencing technologies

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High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing

10.1101/673251 ◽

2019 ◽

Cited By ~ 1

Author(s):

Devika Ganesamoorthy ◽

Mengjia Yan ◽

Valentine Murigneux ◽

Chenxi Zhou ◽

Minh Duc Cao ◽

...

Keyword(s):

Large Scale ◽

Tandem Repeats ◽

Gc Content ◽

Genomic Variation ◽

Population Variation ◽

Small Subset ◽

Sequence Coverage ◽

Copy Numbers ◽

Long Read ◽

Highly Correlated

ABSTRACTTandem repeats (TRs) are highly prone to variation in copy numbers due to their repetitive and unstable nature, which makes them a major source of genomic variation between individuals. However, population variation of TRs have not been widely explored due to the limitations of existing tools, which are either low-throughput or restricted to a small subset of TRs. Here, we used SureSelect targeted sequencing approach combined with Nanopore sequencing to overcome these limitations. We achieved an average of 3062-fold target enrichment on a panel of 142 TR loci, generating an average of 97X sequence coverage on 7 samples utilizing 2 MinION flow-cells with 200ng of input DNA per sample. We identified a subset of 110 TR loci with length less than 2kb, and GC content greater than 25% for which we achieved an average genotyping rate of 75% and increasing to 91% for the highest-coverage sample. Alleles estimated from targeted long-read sequencing were concordant with gold standard PCR sizing analysis and moreover highly correlated with alleles estimated from whole genome long-read sequencing. We demonstrate a targeted long-read sequencing approach that enables simultaneous analysis of hundreds of TRs and accuracy is comparable to PCR sizing analysis. Our approach is feasible to scale for more targets and more samples facilitating large-scale analysis of TRs.

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Robust detection of tandem repeat expansions from long DNA reads

10.1101/356931 ◽

2018 ◽

Cited By ~ 1

Author(s):

Satomi Mitsuhashi ◽

Martin C Frith ◽

Takeshi Mizuguchi ◽

Satoko Miyatake ◽

Tomoko Toyota ◽

...

Keyword(s):

Tandem Repeat ◽

Tandem Repeats ◽

Genetic Diseases ◽

Error Rates ◽

Robust Detection ◽

Sequencing Errors ◽

Tandem Repeat Sequences ◽

Long Read ◽

Repeat Expansions ◽

The Many

AbstractTandemly repeated sequences are highly mutable and variable features of genomes. Tandem repeat expansions are responsible for a growing list of human diseases, even though it is hard to determine tandem repeat sequences with current DNA sequencing technology. Recent long-read technologies are promising, because the DNA reads are often longer than the repetitive regions, but are hampered by high error rates. Here, we report robust detection of human repeat expansions from careful alignments of long (PacBio and nanopore) reads to a reference genome. Our method (tandem-genotypes) is robust to systematic sequencing errors, inexact repeats with fuzzy boundaries, and low sequencing coverage. By comparing to healthy controls, we can prioritize pathological expansions within the top 10 out of 700000 tandem repeats in the genome. This may help to elucidate the many genetic diseases whose causes remain unknown.

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Induction of Recombinant Lectin Expression by an Artificially Constructed Tandem Repeat Structure: A Case Study Using Bryopsis plumosa Mannose-Binding Lectin

Biomolecules ◽

10.3390/biom8040146 ◽

2018 ◽

Vol 8 (4) ◽

pp. 146 ◽

Cited By ~ 3

Author(s):

Hyun-Ju Hwang ◽

Jin-Woo Han ◽

Hancheol Jeon ◽

Jong Han

Keyword(s):

Tandem Repeat ◽

Large Scale ◽

Tandem Repeats ◽

Expression System ◽

Mannose Binding Lectin ◽

Repeat Structure ◽

Tandem Repeat Sequences ◽

Mannose Binding ◽

Bryopsis Plumosa ◽

Binding Lectin

Lectin is an important protein in medical and pharmacological applications. Impurities in lectin derived from natural sources and the generation of inactive proteins by recombinant technology are major obstacles for the use of lectins. Expressing recombinant lectin with a tandem repeat structure can potentially overcome these problems, but few studies have systematically examined this possibility. This was investigated in the present study using three distinct forms of recombinant mannose-binding lectin from Bryopsis plumosa (BPL2)—i.e., the monomer (rD1BPL2), as well as the dimer (rD2BPL2), and tetramer (rD4BPL2) arranged as tandem repeats. The concentration of the inducer molecule isopropyl β-D-1-thiogalactopyranoside and the induction time had no effect on the efficiency of the expression of each construct. Of the tested constructs, only rD4BPL2 showed hemagglutination activity towards horse erythrocytes; the activity of towards the former was 64 times higher than that of native BPL2. Recombinant and native BPL2 showed differences in carbohydrate specificity; the activity of rD4BPL2 was inhibited by the glycoprotein fetuin, whereas that of native BPL2 was also inhibited by d-mannose. Our results indicate that expression as tandem repeat sequences can increase the efficiency of lectin production on a large scale using a bacterial expression system.

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The first genome assembly of fungal pathogen Pyrenophora tritici-repentis race 1 isolate using Oxford Nanopore MinION sequencing

BMC Research Notes ◽

10.1186/s13104-021-05751-0 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Paula Moolhuijzen ◽

Pao Theen See ◽

Caroline S. Moffat

Keyword(s):

Genome Assembly ◽

Fungal Pathogen ◽

Large Scale ◽

Gc Content ◽

Tan Spot ◽

Short Read ◽

Short Reads ◽

Pyrenophora Tritici Repentis ◽

Oxford Nanopore ◽

Long Read

Abstract Objectives The assembly of fungal genomes using short-reads is challenged by long repetitive and low GC regions. However, long-read sequencing technologies, such as PacBio and Oxford Nanopore, are able to overcome many problematic regions, thereby providing an opportunity to improve fragmented genome assemblies derived from short reads only. Here, a necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) isolate 134 (Ptr134), which causes tan spot disease on wheat, was sequenced on a MinION using Oxford Nanopore Technologies (ONT), to improve on a previous Illumina short-read genome assembly and provide a more complete genome resource for pan-genomic analyses of Ptr. Results The genome of Ptr134 sequenced on a MinION using ONT was assembled into 28 contiguous sequences with a total length of 40.79 Mb and GC content of 50.81%. The long-read assembly provided 6.79 Mb of new sequence and 2846 extra annotated protein coding genes as compared to the previous short-read assembly. This improved genome sequence represents near complete chromosomes, an important resource for large scale and pan genomic comparative analyses.

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High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell

10.1101/149997 ◽

2017 ◽

Cited By ~ 7

Author(s):

Todd P. Michael ◽

Florian Jupe ◽

Felix Bemm ◽

S. Timothy Motley ◽

Justin P. Sandoval ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Large Scale ◽

Cost Effective ◽

New Era ◽

Genome Maps ◽

Oxford Nanopore ◽

Long Read ◽

Genome Assemblies ◽

Typical Consumer ◽

Arabidopsis Thaliana Genome

AbstractWhile many evolutionary questions can be answered by short read re-sequencing, presence/absence polymorphisms of genes and/or transposons have been largely ignored in large-scale intraspecific evolutionary studies. To enable the rigorous analysis of such variants, multiple high quality and contiguous genome assemblies are essential. Similarly, while genome assemblies based on short reads have made genomics accessible for non-reference species, these assemblies have limitations due to low contiguity. Long-read sequencers and long-read technologies have ushered in a new era of genome sequencing where the lengths of reads exceed those of most repeats. However, because these technologies are not only costly, but also time and compute intensive, it has been unclear how scalable they are. Here we demonstrate a fast and cost effective reference assembly for an Arabidopsis thaliana accession using the USB-sized Oxford Nanopore MinION sequencer and typical consumer computing hardware (4 Cores, 16Gb RAM). We assemble the accession KBS-Mac-74 into 62 contigs with an N50 length of 12.3 Mb covering 100% (119 Mb) of the non-repetitive genome. We demonstrate that the polished KBS-Mac-74 assembly is highly contiguous with BioNano optical genome maps, and of high per-base quality against a likewise polished Pacific Biosciences long-read assembly. The approach we implemented took a total of four days at a cost of less than 1,000 USD for sequencing consumables including instrument depreciation.

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An Integrated Sequencing Approach for Updating of Pseudorabies Virus Transcriptome

10.20944/preprints202012.0616.v1 ◽

2020 ◽

Author(s):

Gábor Torma ◽

Dóra Tombácz ◽

Zsolt Csabai ◽

Dániel Göbhardter ◽

Zoltán Deim ◽

...

Keyword(s):

Pseudorabies Virus ◽

Large Fraction ◽

Viral Gene Expression ◽

Transcriptome Profiling ◽

Current Data ◽

Viral Gene ◽

Systematic Analysis ◽

Rna Molecules ◽

Oxford Nanopore ◽

Long Read

In the last couple of years, the implementation of long-read sequencing (LRS) technologies for transcriptome profiling has uncovered an extreme complexity of viral gene expression. In this study, we carried out a systematic analysis on the pseudorabies virus transcriptome by combining our current data obtained by using Pacific Biosciences Sequel and Oxford Nanopore Technologies MinION sequencings with our earlier data generated by other LRS and short-read sequencing techniques. As a result, we identified a number of novel genes, transcripts, and transcript isoforms, including splice and length variants, and also confirmed earlier annotated RNA molecules. One of the major findings of this study is the discovery of a large number of 5’-truncated putative mRNAs embedded into larger host mRNAs. A large fraction of these RNA molecules contain in-frame ORFs, which may encode N-terminally truncated polypeptides. These study demonstrates that the PRV transcriptome is much more complex than previously appreciated.

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GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing

10.1101/246108 ◽

2018 ◽

Cited By ~ 1

Author(s):

Devika Ganesamoorthy ◽

Minh Duc Cao ◽

Tania Duarte ◽

Wenhan Chen ◽

Lachlan Coin

Keyword(s):

High Throughput ◽

Tandem Repeat ◽

Copy Number ◽

Tandem Repeats ◽

High Throughput Sequencing ◽

Sequence Data ◽

Complex Diseases ◽

Sequencing Analysis ◽

Reference Dataset ◽

Long Read

ABSTRACTBackgroundTandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations.MethodsWe report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely – GtTR - which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation.ResultsWe used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68% and 83% for capture sequence data and 200X WGS data respectively, improving to 87% and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25%, 14%, 12% and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results.ConclusionsThe novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.

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