scholarly journals Reducing the Number of Mismatches between Hairs and Buccal References When Analysing mtDNA Heteroplasmic Variation by Massively Parallel Sequencing

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1355
Author(s):  
Kristiaan J. van der Gaag ◽  
Stijn Desmyter ◽  
Sophie Smit ◽  
Lourdes Prieto ◽  
Titia Sijen
Keyword(s):  
Sanger Sequencing ◽  
Nuclear Dna ◽  
Massively Parallel Sequencing ◽  
Detection Threshold ◽  
Hair Shaft ◽  
Massively Parallel ◽  
Sequencing Data ◽  
Parallel Sequencing ◽  
Low Level ◽  
Minor Variant

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed.

scholarly journals Noninvasive Prenatal Diagnosis of Monogenic Diseases by Targeted Massively Parallel Sequencing of Maternal Plasma: Application to β-Thalassemia

2012 ◽  
Vol 58 (10) ◽  
pp. 1467-1475 ◽  
Author(s):  
Kwan-Wood G Lam ◽  
Peiyong Jiang ◽  
Gary J W Liao ◽  
K C Allen Chan ◽  
Tak Y Leung ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Massively Parallel Sequencing ◽  
Globin Gene ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Sequencing Data ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Noninvasive Prenatal Diagnosis ◽  
Monogenic Diseases

Abstract BACKGROUND A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.

scholarly journals SICTIN: Rapid footprinting of massively parallel sequencing data

2010 ◽  
Vol 3 (1) ◽  
Author(s):  
Stefan Enroth ◽  
Robin Andersson ◽  
Claes Wadelius ◽  
Jan Komorowski
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Sequencing Data ◽  
Parallel Sequencing

scholarly journals Detection of CYP2C19 Genetic Variants in Malaysian Orang Asli from Massively Parallel Sequencing Data

PLoS ONE ◽  
2016 ◽  
Vol 11 (10) ◽  
pp. e0164169 ◽  
Author(s):  
Geik Yong Ang ◽  
Choo Yee Yu ◽  
Vinothini Subramaniam ◽  
Mohd Ikhmal Hanif Abdul Khalid ◽  
Tuan Azlin Tuan Abdu Aziz ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Sequencing Data ◽  
Parallel Sequencing ◽  
Orang Asli

scholarly journals Laboratory Verification of a BRCA1 and BRCA2 Massively Parallel Sequencing Assay from Wet Bench to Bioinformatics for Germline DNA Analysis

2021 ◽  
Author(s):  
Kok-Siong Poon ◽  
Lily Chiu ◽  
Karen Mei-Ling Tan
Keyword(s):  
Genetic Testing ◽  
Sanger Sequencing ◽  
Dna Analysis ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Brca1 And Brca2 ◽  
Clinical Diagnostic Laboratory ◽  
Parallel Sequencing ◽  
Miseq Platform ◽  
Brca Genetic Testing

Abstract Introduction A robust genetic test for BRCA1 and BRCA2 genes is necessary for the diagnosis, prognosis, and treatment of patients with hereditary breast and ovarian cancer. We evaluated a commercial amplicon-based massively parallel sequencing (MPS) assay, BRCA MASTR Plus on the MiSeq platform, for germline BRCA genetic testing. Methods This study was performed on 31 DNA from cell lines and proficiency testing samples to establish the accuracy of the assay. A reference cell line DNA, NA12878 was used to determine the reproducibility of the assay. Discordant MPS result was resolved orthogonally by the current gold-standard Sanger sequencing method. Results The analytical accuracy, sensitivity, and specificity for variant detection were 93.55, 92.86, and 100.00%, respectively. Both sequencing depth and variant allele frequencies were highly reproducible by comparing the NA12878 DNA tested in three separate runs. The single discordant result, later confirmed by Sanger sequencing was due to the inability of the MASTR Reporter software to identify a 40-bp deletion in BRCA1. Conclusion The BRCA MASTR Plus assay on the MiSeq platform is accurate and reproducible for germline BRCA genetic testing, making it suitable for use in a clinical diagnostic laboratory. However, Sanger sequencing may still serve as a confirmatory method to improve diagnostic capability of the MPS assay.

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