Validation of a Long-Read PCR Assay for Sensitive Detection and Sizing of C9orf72 Hexanucleotide Repeat Expansions

Suh, EunRan; Grando, Kaitlyn; Deerlin, Vivianna M. Van

doi:10.1016/j.jmoldx.2018.07.001

Cited by 12 publications

(10 citation statements)

References 29 publications

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“…We discovered 3 distinct read-length populations in each sample, potentially representing significant mosaicism. This observation is not uncommon in studies of repeat expansions where genotyping assays are performed on cell line-derived DNA (38,39). Determining whether these 3 alleles were present in the blood of these patients or arose as an artifact of cell culture or sequencing would require both blood and LCL-derived DNA from the same individual, which is not available for the NINDS ALS Collection.…”

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: Discussionmentioning

confidence: 99%

“…fragments are more likely to be damaged between the flanking Cas9 binding sites, which would result in failure of enrichment. The presence of the three alleles in each sample were confirmed by repeated via Southern blot during validation of a PCR-based assay (38).…”

Section: Cabage Target Enrichment Produces Reads That Span a Pathogenmentioning

confidence: 99%

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

Wallace

Sasani

Swanier

et al. 2020

Preprint

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“…Normal repeat length was defined as ≤28 repeats. Samples revealing a single peak product were further analyzed by long-read PCR using the AmplideX PCR/CE C9orf72 (RUO) Assay (Asuragen, Inc.) as previously described (Suh et al, 2018). Amplidex PCR technique uses gene-specific and repeat-specific primers and provides an accurate capillary electrophoresis sizing of alleles up to 145 GGGGCC repeats and identifies the presence of expanded alleles over 145 repeats.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive Genetic Analysis of a Hungarian Amyotrophic Lateral Sclerosis Cohort

et al. 2019

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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the degeneration of motor neurons. Genetic factors play a key role in ALS, and identifying variants that contribute to ALS susceptibility is an important step toward understanding the etiology of the disease. The frequency of protein altering variants in ALS patients has been extensively investigated in populations of different ethnic origin. To further delineate the genetic architecture of the Hungarian ALS patients, we aimed to detect potentially damaging variants in major and minor ALS genes and in genes related to other neurogenetic disorders. A combination of repeat-sizing of C9orf72 and next-generation sequencing (NGS) was used to comprehensively assess genetic variations in 107 Hungarian patients with ALS. Variants in major ALS genes were detected in 36.45% of patients. As a result of repeat sizing, pathogenic repeat expansions in the C9orf72 gene were detected in 10 patients (9.3%). According to the NGS results, the most frequently mutated genes were NEK1 (5.6%), NEFH , SQSTM1 (3.7%), KIF5A , SPG11 (2.8%), ALS2 , CCNF , FUS , MATR3 , TBK1 , and UBQLN2 (1.9%). Furthermore, potentially pathogenic variants were found in GRN and SIGMAR1 genes in single patients. Additional 33 novel or rare known variants were detected in minor ALS genes, as well as 48 variants in genes previously linked to other neurogenetic disorders. The latter finding supports the hypothesis that common pathways in different neurodegenerative diseases may contribute to the development of ALS. While the disease-causing role of several variants identified in this study has previously been established, other variants may show reduced penetrance or may be rare benign variants. Our findings highlight the necessity for large-scale multicenter studies on ALS patients to gain a more accurate view of the genetic pattern of ALS.

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“…The appearance of the third alleles in these samples could be artifacts of cell line transformation from which the DNA was derived. Multiple populations of allele lengths have been previously observed in cell lines and was observed in ND11836 via Southern blot during validation of a PCR-based assay [ 44 ].…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2021

View full text Add to dashboard Cite

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 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 a median of 116X coverage (range 39–416) 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.

show abstract

Validation of a Long-Read PCR Assay for Sensitive Detection and Sizing of C9orf72 Hexanucleotide Repeat Expansions

Cited by 12 publications

References 29 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

Comprehensive Genetic Analysis of a Hungarian Amyotrophic Lateral Sclerosis Cohort

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

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