A mixing heteroduplex mobility assay (mHMA) to genotype homozygous mutants with small indels generated by CRISPR-Cas9 nucleases

Rice is a salt-sensitive species with enormous genetic variation for salt tolerance hidden in its germplasm pool. The EcoTILLING technique allows us to assign haplotypes, thus reducing the number of accessions to be sequenced, becoming a cost-effective, time-saving and high-throughput method, ideal to be used in laboratories with limited financial resources. Aiming to find alleles associated with salinity tolerance, we are currently using the EcoTILLING technique to detect single nucleotide polymorphisms (SNPs) and small indels across 375 germplasm accessions representing the diversity available in domesticated rice. We are targeting several genes known to be involved in salt stress signal transduction (OsCPK17) or tolerance mechanisms (SalT). So far, we found a total of 15 and 23 representative SNPs or indels in OsCPK17 and SalT, respectively. These natural allelic variants are mostly located in 3′-untranslated region, thus opening a new path for studying their potential contribution to the regulation of gene expression and possible role in salt tolerance.

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Investigation of varicella-zoster virus variation by heteroduplex mobility assay

Immunity to and Prevention of Herpes Zoster ◽

10.1007/978-3-7091-6259-0_3 ◽

2001 ◽

pp. 17-25 ◽

Cited By ~ 6

Author(s):

W. Barrett-Muir ◽

K. Hawrami ◽

J. Clarke ◽

J. Breuer

Keyword(s):

Varicella Zoster Virus ◽

Varicella Zoster ◽

Heteroduplex Mobility Assay ◽

Virus Variation

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Single-tube genotyping for small insertion/deletion mutations: simultaneous identification of wild type, mutant and heterozygous alleles

Biology Methods and Protocols ◽

10.1093/biomethods/bpaa007 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Bin Lin ◽

Jing Sun ◽

Iain D C Fraser

Keyword(s):

Wild Type ◽

Single Polymerase Chain Reaction ◽

Deletion Mutations ◽

Systematic Testing ◽

Small Indels ◽

Naturally Occurring ◽

Induced Mutagenesis ◽

Type Mutant ◽

Polymerase Chain ◽

Simultaneous Identification

Abstract Current methods of genotyping small insertion/deletion (indel) mutations are costly, laborious, and can be unreliable. To address this, we have developed a method for small indel genotyping in a single polymerase chain reaction, with wild-type, heterozygous and mutant alleles distinguishable by band pattern in routine agarose gel electrophoresis. We demonstrate this method with multiple genes to distinguish 10 bp, 4 bp and even 1 bp deletions from the wild type. Through systematic testing of numerous primer designs, we also propose guidelines for genotyping small indel mutations. Our method provides a convenient approach to genotyping small indels derived from clustered regularly interspaced short palindromic repeats-mediated gene editing, N-ethyl-N-nitrosourea induced mutagenesis or diagnosis of naturally occurring polymorphisms/mutations.

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Heteroduplex mobility assay for rapid, sensitive and specific detection of mycobacteria

Diagnostic Microbiology and Infectious Disease ◽

10.1016/s0732-8893(00)00112-7 ◽

2000 ◽

Vol 36 (4) ◽

pp. 225-235 ◽

Cited By ~ 6

Author(s):

Agdemir Waléria-Aleixo ◽

Erna G Kroon ◽

Marco A.S Campos ◽

Maria E Margutti–Pinto ◽

Claudio A Bonjardim ◽

...

Keyword(s):

Specific Detection ◽

Heteroduplex Mobility Assay

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Genetic Diversity of Pseudomonas syringae Pathovars and Related Species Assessed by DNA Heteroduplex Mobility Assay

Plant Pathogenic Bacteria ◽

10.1007/978-94-010-0003-1_25 ◽

2001 ◽

pp. 128-130 ◽

Cited By ~ 1

Author(s):

L. Sutra ◽

P. Simoneau ◽

L. Gardan

Keyword(s):

Genetic Diversity ◽

Pseudomonas Syringae ◽

Related Species ◽

Heteroduplex Mobility Assay ◽

Pseudomonas Syringae Pathovars

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Development and Validation of a Next-Generation Sequencing Panel for Syndromic and Nonsyndromic Hearing Loss

The Journal of Applied Laboratory Medicine ◽

10.1093/jalm/jfaa021 ◽

2020 ◽

Vol 5 (3) ◽

pp. 467-479 ◽

Cited By ~ 1

Author(s):

Malinda Butz ◽

Amber McDonald ◽

Patrick A Lundquist ◽

Melanie Meyer ◽

Sean Harrington ◽

...

Keyword(s):

Hearing Loss ◽

Next Generation Sequencing ◽

Copy Number Variants ◽

Analytical Sensitivity ◽

Nonsyndromic Hearing Loss ◽

Next Generation ◽

Single Nucleotide Variants ◽

Small Indels ◽

Syndromic Hearing Loss ◽

Generation Sequencing

Abstract Background Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. Methods We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. Results We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1–22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. Conclusions We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

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Whole exome sequencing and copy-number variation analysis of 20 NF2-associated spinal and cranial meningiomas.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.2051 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 2051-2051

Author(s):

Ramita Dewan ◽

Alexander Pemov ◽

Nancy F Hansen ◽

Settara C Chandrasekharappa ◽

Jim Mullikin ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Snp Array ◽

Neurofibromatosis Type ◽

Large Chromosome ◽

Nucleotide Substitutions ◽

Genetic Event ◽

Multiple Tumor ◽

Small Indels ◽

Whole Exome

2051 Background: Neurofibromatosis type 2 is a heritable tumor predisposition syndrome characterized by the growth of multiple tumor types in the nervous system, including bilateral vestibular schwannomas, meningiomas and ependymomas. Recent genomic sequencing studies have revealed that NF2 inactivation is the most frequent genetic event in sporadic meningiomas. In line with Knudson’s hypothesis, it is accepted that somatic inactivation of the wildtype NF2 allele initiates tumor growth in NF2 patients, but little is known of what other genes or pathways influence meningioma tumorigenesis. Methods: To investigate this question, we performed whole exome sequencing (WES) (Illumina Hi-Seq 2500 platform, 96 Mb SeqCap EZ Exome + UTR Library, NimbleGen) and SNP-array analysis (HumanOmniExpressExome-8, v1.2 arrays, Illumina) of twenty spinal and cranial meningioma samples from seven NF2 patients. Mutation validation was completed via orthogonal sequencing (IonTorrent, ThermoFisher). Results: We identified NF2 germline mutations in all patients, including five nonsense, one splice site and one likely pathogenic intronic mutation. We found that the predominant mechanism of somatic NF2 inactivation in the tumors was loss of heterozygosity (LOH): we identified large chromosome 22 deletions containing NF2 in nineteen out of twenty meningiomas. The second most frequent chromosomal aberration was a deletion within chromosome 1p, followed by entire chromosome X deletion and rearrangements in chromosome 17q. The remaining samples exhibited normal diploid genomic architecture. Somatic mutations included about twenty point substitutions and small indels. Conclusions: Our study revealed that somatic inactivation of NF2 is the most frequent and only recurrent genetic event in NF2-associated meningiomas. Large LOH events are the most prevalent second hit mechanism and may also represent a common path of meningioma progression. Somatic single nucleotide substitutions and small indels are rare in these tumors. Interestingly, we did not identify mutations in TRAF7, KLF4, AKT1, or SMO, which have been found to be critical for non-NF2 meningioma growth.

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MET exon 14 skipping analogs: Rare but potentially clinically actionable.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.3141 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 3141-3141 ◽

Cited By ~ 1

Author(s):

Rebecca Feldman ◽

Michelle Ellis ◽

Jeffrey Swensen ◽

Wolfgang Michael Korn ◽

Zoran Gatalica

Keyword(s):

Receptor Gene ◽

Negative Regulation ◽

Clinicopathological Features ◽

Smoking History ◽

Protein Overexpression ◽

Juxtamembrane Domain ◽

Oncogenic Signaling ◽

Small Indels ◽

Nsclc Patients ◽

The Impact

3141 Background: The DpY motif within the exon 14 juxtamembrane domain of the MET receptor gene is critical for Cbl-mediated negative regulation. Splicing alterations that delete this residue, known as exon 14 skipping mutations (ex14sk mt), lead to prolonged MET protein stability and oncogenic signaling. Specific mt at the Y1021 (aka 1003) residue are thought to lead to similar effects as ex14sk, but due to their rarity, their role in NSCLC is unknown. We sought to identify and characterize non-ex14sk mt that include/surround Y1021. Methods: Retrospective review of molecular profiles for non-ex14sk mt that include/surround the DpY motif (Y1021) in MET. Two NGS platforms were included: MiSeq (2014-2017; n=2865) and NextSeq (2017-2019; n=6084). Immunohistochemistry (IHC) of cMET (SP44) and co-occurring alterations (EGFR, KRAS, ALK, ROS) were also reviewed. Results: Of 8,949 NSCLC patients with successful NGS of MET gene by either platform, 13 cases or 0.2% were identified to have an alteration within the amino acids of interest. Eleven cases included substitutions at Y1021 (5 phenylalanine, 4 histidine and 3 arginine) and the remaining two cases included small insertion-deletions p.E1017_Y1021delinsH and p.D1020_Y1021delinsV, the latter was later excluded as it co-harbored an ex14sk mt. Conclusions: Similar to patients with ex14sk mt, substitutions and small indels at Y1021 exhibit Clinicopathological features such as previous smoking history and older age, mutual exclusivity with oncogene drivers and MET protein overexpression. The rarity of these analogous ex14sk mt suggests deletions of exon 14 provide cellular advantages beyond Cbl-mediated ubiquitinylation of MET. Although rare, the impact of these mt on efficacy of Met-directed therapy deserves further exploration.[Table: see text]

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