scholarly journals Performance Characterization and Validation of Saliva as an Alternative Specimen Source for Detecting Hereditary Breast Cancer Mutations by Next Generation Sequencing

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Varsha Meghnani ◽  
Nadeem Mohammed ◽  
Christopher Giauque ◽  
Rahul Nahire ◽  
Thomas David
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Germline Mutations ◽  
Quality Parameters ◽  
Common Source ◽  
Sample Type ◽  
Next Generation ◽  
Alternative Source ◽  
Cancer Mutations ◽  
Generation Sequencing

Identification of pathogenic germline mutations by next generation sequencing is a widely accepted tool for predicting the risk of hereditary cancer development. Blood is the most common source of DNA for such tests. However, blood as a sample type has many drawbacks, including the invasive collection method, poor sample stability, and a relatively high cost of collection. Therefore, in the current study we have assessed the suitability of saliva as an alternative source of genomic DNA for the identification of germline mutations in the BRCA1/2 genes by next generation sequencing (NGS). Our results show that all of the samples yielded DNA concentrations sufficient for library preparation. The concentrations of the final libraries, which were generated by PCR using target specific primers, fall into the expected range with no notable difference between libraries generated from DNA derived from saliva or blood. Quality parameters indicate that sequencing performance is comparable across sample source. An average of (98±0.02)% variant calling concordance was obtained between the two specimen sources. Our data recommends saliva as a potential alternative for detecting germline mutation by next generation sequencing.

scholarly journals Improved next-generation sequencing pre-capture library yields and sequencing parameters using on-bead PCR

BioTechniques ◽  
2020 ◽  
Vol 68 (1) ◽  
pp. 48-51 ◽  
Author(s):  
Christopher R McEvoy ◽  
Timothy Semple ◽  
Bhargavi Yellapu ◽  
David Y Choong ◽  
Huiling Xu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Limiting Factor ◽  
Next Generation ◽  
Tumor Biopsy ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna ◽  
Generation Sequencing

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor.

Spectrum of Pathogenic Germline Mutations in Chinese Lung Cancer Patients through Next-Generation Sequencing

2019 ◽  
Vol 26 (1) ◽  
pp. 109-114 ◽  
Author(s):  
Panwen Tian ◽  
Xiangyang Cheng ◽  
Zhengyi Zhao ◽  
Yuzi Zhang ◽  
Celimuge Bao ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Cancer Patients ◽  
Germline Mutations ◽  
Next Generation ◽  
Lung Cancer Patients ◽  
Generation Sequencing

scholarly journals Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6661 ◽  
Author(s):  
Arianna Nicolussi ◽  
Francesca Belardinilli ◽  
Yasaman Mahdavian ◽  
Valeria Colicchia ◽  
Sonia D’Inzeo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Germline Mutations ◽  
Ion Torrent ◽  
Next Generation ◽  
Training Set ◽  
Brca1 And Brca2 ◽  
Ion Torrent Pgm ◽  
Validation Set ◽  
Generation Sequencing

Background Conventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment. Methods We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set). Results By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis. Discussion Our experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.

scholarly journals Detection of BRCA1 and BRCA2 germline mutations in Japanese population using next‐generation sequencing

2014 ◽  
Vol 3 (2) ◽  
pp. 121-129 ◽  
Author(s):  
Yosuke Hirotsu ◽  
Hiroshi Nakagomi ◽  
Ikuko Sakamoto ◽  
Kenji Amemiya ◽  
Hitoshi Mochizuki ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Japanese Population ◽  
Germline Mutations ◽  
Next Generation ◽  
Brca1 And Brca2 ◽  
Generation Sequencing

scholarly journals A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiandong Zeng ◽  
Natalia T. Leach ◽  
Zhaoqing Zhou ◽  
Hui Zhu ◽  
Jean A. Smith ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Causative Mutation ◽  
Next Generation ◽  
Single Nucleotide ◽  
Highly Sensitive ◽  
Highly Sensitive Detection ◽  
Next Generation Sequencing Ngs ◽  
Reference Bias ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

scholarly journals Next-Generation Sequencing in Order to Better Characterize a BRCA Variant of Uncertain Significance

2017 ◽  
Vol 10 (2) ◽  
pp. 634-637 ◽  
Author(s):  
Steven Sorscher ◽  
Shakti Ramkissoon
Keyword(s):  
Next Generation Sequencing ◽  
Germline Mutations ◽  
Allelic Frequency ◽  
Breast Cancers ◽  
Next Generation ◽  
Cancer Syndrome ◽  
Predisposing Factor ◽  
Variant Of Uncertain Significance ◽  
Uncertain Significance ◽  
Generation Sequencing

BRCA germline mutations are the most common predisposing factor in familial breast-ovarian cancer syndrome families. However, many screened patients are identified as harboring BRCA variants of uncertain significance (VUS), rather than carrying deleterious germline mutations [Calo et al.: Cancers 2010; 2:1644–1660]. While such VUSs are typically reclassified as benign polymorphisms, this may occur years after the VUS is first identified [Murray et al.: Genet Med 2011; 13; 998–1005]. Loss of heterozygosity (LOH) of BRCA is nearly always the gatekeeper event in inherited BRCA-related breast cancer and LOH of BRCA is rare in sporadic cancers [Osorio et al.: Int J Cancer 2002; 99:305–309]. Here, we describe a patient identified as carrying a germline BRCA VUS. Tumor next-generation sequencing (NGS) demonstrated a very high mutation allelic frequency for that BRCA VUS, consistent with LOH. This case illustrates that since BRCA LOH is the typical mechanism of transformation in inherited BRCA-related breast cancers, NGS might be used to suggest that the BRCA VUS is actually cancer predisposing in a particular family. As a result, this may help patients make more informed decisions regarding screening and prophylactic therapy, long before official reclassification of the VUS occurs.

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