Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing

Gießelmann, Pay; Brändl, Björn; Raimondeau, Etienne; Bowen, Rebecca; Rohrandt, Christian; Tandon, R. P.; Kretzmer, Helene; Assum, Günter; Galonska, Christina; Siebert, Reiner; Ammerpohl, Ole; Heron, Andrew J.; Schneider, Susanne A.; Ladewig, Julia; Koch, Philipp; Schuldt, Bernhard; Graham, James E.; Meissner, Alexander; Müller, Franz‐Josef

doi:10.1038/s41587-019-0293-x

Cited by 134 publications

(154 citation statements)

References 41 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: 80%

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: 80%

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

show abstract

“…For example, the minisatellite length polymorphisms at the insulin gene VNTR is strongly associated with juvenile obesity and metabolic syndrome (46). Expansions of short tandem repeats (e.g., GGGGCC repeats within C9orf72 gene in amyotrophic lateral sclerosis and CGG repeats within FMR1 gene in fragile X syndrome) are genetic variants that have been implicated in several neuropsychiatric and other disorders, but their assessment remains challenging with Southern blot-based method (47). We envision the principles of STAR assay to be extended for many of these applications for high-throughput screening to replace current cumbersome gel electrophoresis procedures.…”

Section: Discussionmentioning

confidence: 99%

Massively parallel single-molecule telomere length measurement with digital real-time PCR

et al. 2020

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Telomere length is a promising biomarker for age-associated diseases and cancer, but there are still substantial challenges to routine telomere analysis in clinics because of the lack of a simple and rapid yet scalable method for measurement. We developed the single telomere absolute-length rapid (STAR) assay, a novel high-throughput digital real-time PCR approach for rapidly measuring the absolute lengths and quantities of individual telomere molecules. We show that this technique provides the accuracy and sensitivity to uncover associations between telomere length distribution and telomere maintenance mechanisms in cancer cell lines and primary tumors. The results indicate that the STAR assay is a powerful tool to enable the use of telomere length distribution as a biomarker in disease and population-wide studies.

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“…We were interested in studying FMR1 at a singlemolecule level because of the link between the epigenetic status of the FMR1 promoter and the number of CCG repeats, and since repeat number and methylation mosaicism are common phenomena in FXS. Analysis of these genetic and epigenetic features on native molecules of FMR1 has been attempted using both SMRT sequencing and nanopore sequencing but in neither case was it possible to perform accurate repeat sizing and methylation analysis on the same single molecules 48,49 . Our MT platform is well suited to this dual analysis in the same single molecules of DNA.…”

Section: Discussionmentioning

confidence: 99%

Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform

Wang¹,

Maluenda²,

Giraut³

et al. 2020

Preprint

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Accurate decoding of nucleic acid variation is important to understand the complexity and regulation of genome function. Here we introduce a single-molecule platform based on magnetic tweezer (MT) technology that can identify and map the positions of sequence variation and multiple base modifications together in the same single molecules of DNA or RNA at single base resolution. Using synthetic templates, we demonstrate that our method can distinguish the most common epigenetic marks on DNA and RNA with high sensitivity, specificity and precision. We also developed a highly specific CRISPR-Cas enrichment strategy to target genomic regions in native DNA without amplification. We then used this method to enrich native DNA from E. coli and characterized the differential levels of adenine and cytosine base modifications together in molecules of up to 5 kb in length. Finally, we enriched the 5'UTR of FMR1 from cells derived from a Fragile X carrier and precisely measured the repeat expansion length and methylation status of each molecule. These results demonstrate that our platform can detect a variety of genetic, epigenetic and base modification changes concomitantly within the same single molecules.

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Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing

Cited by 134 publications

References 41 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

Massively parallel single-molecule telomere length measurement with digital real-time PCR

Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform

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