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

Roeck, Arne De; Coster, Wouter De; Bossaerts, Liene; Cacace, Rita; Pooter, Tim De; Dongen, Jasper Van; D’Hert, Svenn; Rijk, Peter De; Stražišar, Mojca; Broeckhoven, Christine Van; Sleegers, Kristel

doi:10.1186/s13059-019-1856-3

Cited by 58 publications

(41 citation statements)

References 39 publications

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“…Further, both samples showed a strong bias to sequencing the shortest allele, representing 79% and 91% of the spanning reads, respectively. This is likely an artifact of the technology sequencing shorter fragments more efficiently, as has been previously observed (35)(36)(37) and the fact that longer (e.g. expanded)…”

Section: Cabage Target Enrichment Produces Reads That Span a Pathogenmentioning

confidence: 83%

“…We note that several reads representing the expanded alleles failed base calling using Guppy and were retrieved from the "fastq_fail" folder generated by the MinKNOW software. Nanosatellite is specifically designed software to accurately characterize tandem repeat sequences where standard base calling algorithms perform poorly (35) Guide RNA design. Guide RNAs (sgRNA, S1 Table) were selected to flank up and downstream of the Sequence data alignment and QC.…”

Section: Discussionmentioning

confidence: 99%

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CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

Wallace

Sasani

Swanier

et al. 2020

Preprint

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A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore’s MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a novel method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore’s MinION long-read sequencing technology. Enrichment with CaBagE resulted in up to 416X coverage of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the ‘hidden genome’ underlying human disease.

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Section: Cabage Target Enrichment Produces Reads That Span a Pathogenmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

Wallace

Sasani

Swanier

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Hundreds of thousands of individuals' genomes have now been sequenced using short-read sequencing from many large disease cohorts, awaiting additional analyses such as RE detection. Additionally, while it is generally easier to analyze the structure of the expanded repeats in long-read data [44,45], combining short-read sequencing datasets and methods with long-read data can offer a cost-effective way to conduct large-scale repeat expansion discovery projects.…”

Section: Discussionmentioning

confidence: 99%

ExpansionHunter Denovo: a computational method for locating known and novel repeat expansions in short-read sequencing data

et al. 2020

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Repeat expansions are responsible for over 40 monogenic disorders, and undoubtedly more pathogenic repeat expansions remain to be discovered. Existing methods for detecting repeat expansions in short-read sequencing data require predefined repeat catalogs. Recent discoveries emphasize the need for methods that do not require pre-specified candidate repeats. To address this need, we introduce ExpansionHunter Denovo, an efficient catalog-free method for genome-wide repeat expansion detection. Analysis of real and simulated data shows that our method can identify large expansions of 41 out of 44 pathogenic repeats, including nine recently reported non-reference repeat expansions not discoverable via existing methods.

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“…A promising example is the development of the R10.3 flow cell, an updated version of the R10 flow cell used in this study [24]. Genotyping based on the raw data, squiggles, has proven its value for large tandem repeats and could potentially be developed for forensic STRs [25].…”

Section: Nanopore Sequencing-based Genotypingmentioning

confidence: 99%

Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling

Tytgat

Gansemans

Weymaere

et al. 2020

Genes

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Nanopore sequencing for forensic short tandem repeats (STR) genotyping comes with the advantages associated with massively parallel sequencing (MPS) without the need for a high up-front device cost, but genotyping is inaccurate, partially due to the occurrence of homopolymers in STR loci. The goal of this study was to apply the latest progress in nanopore sequencing by Oxford Nanopore Technologies in the field of STR genotyping. The experiments were performed using the state of the art R9.4 flow cell and the most recent R10 flow cell, which was specifically designed to improve consensus accuracy of homopolymers. Two single-contributor samples and one mixture sample were genotyped using Illumina sequencing, Nanopore R9.4 sequencing, and Nanopore R10 sequencing. The accuracy of genotyping was comparable for both types of flow cells, although the R10 flow cell provided improved data quality for loci characterized by the presence of homopolymers. We identify locus-dependent characteristics hindering accurate STR genotyping, providing insights for the design of a panel of STR loci suited for nanopore sequencing. Repeat number, the number of different reference alleles for the locus, repeat pattern complexity, flanking region complexity, and the presence of homopolymers are identified as unfavorable locus characteristics. For single-contributor samples and for a limited set of the commonly used STR loci, nanopore sequencing could be applied. However, the technology is not mature enough yet for implementation in routine forensic workflows.

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

Cited by 58 publications

References 39 publications

CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

CaBagE: a Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing

ExpansionHunter Denovo: a computational method for locating known and novel repeat expansions in short-read sequencing data

Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling

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