Targeted Next-Generation Sequencing can Replace Sanger Sequencing in Clinical Diagnostics

2013 ◽  
Vol 34 (7) ◽  
pp. 1035-1042 ◽  
Author(s):  
Birgit Sikkema-Raddatz ◽  
Lennart F. Johansson ◽  
Eddy N. de Boer ◽  
Rowida Almomani ◽  
Ludolf G. Boven ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Clinical Diagnostics ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

scholarly journals Comparison of targeted next-generation sequencing and Sanger sequencing for the detection of PIK3CA mutations in breast cancer

2015 ◽  
Vol 15 (1) ◽  
Author(s):  
Ruza Arsenic ◽  
Denise Treue ◽  
Annika Lehmann ◽  
Michael Hummel ◽  
Manfred Dietel ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Next Generation ◽  
Pik3ca Mutations ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

Identification of a Disease-Causing Mutation in a Chinese Patient with Retinitis Pigmentosa by Targeted Next-Generation Sequencing

2017 ◽  
Vol 27 (6) ◽  
pp. 791-796 ◽  
Author(s):  
Jianping Xiao ◽  
Xueqin Guo ◽  
Yong Wang ◽  
Mingkun Shao ◽  
Xiaoming Wei ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Retinitis Pigmentosa ◽  
Sanger Sequencing ◽  
Bioinformatics Analysis ◽  
Family Members ◽  
Chinese Patient ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Generation Sequencing

Purpose To identify disease-causing mutations in a Chinese patient with retinitis pigmentosa (RP). Methods A detailed clinical examination was performed on the proband. Targeted next-generation sequencing (NGS) combined with bioinformatics analysis was performed on the proband to detect candidate disease-causing mutations. Sanger sequencing was performed on all subjects to confirm the candidate mutations and assess cosegregation within the family. Results Clinical examinations of the proband showed typical characteristics of RP. Three candidate heterozygous mutations in 3 genes associated with RP were detected in the proband by targeted NGS. The 3 mutations were confirmed by Sanger sequencing and the deletion (c.357_358delAA) in PRPF31 was shown to cosegregate with RP phenotype in 7 affected family members, but not in 3 unaffected family members. Conclusions The deletion (c.357_358delAA) in PRPF31 was the disease-causing mutation for the proband and his affected family members with RP. To our knowledge, this is the second report of the deletion and the first report of the other 2 mutations in the Chinese population. Targeted NGS combined with bioinformatics analysis proved to be an effective molecular diagnostic tool for RP.

scholarly journals Molecular diagnosis of inherited peripheral neuropathies by targeted next-generation sequencing: molecular spectrum delineation

BMJ Open ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. e021632 ◽  
Author(s):  
Juliette Bacquet ◽  
Tanya Stojkovic ◽  
Amandine Boyer ◽  
Nathalie Martini ◽  
Frédérique Audic ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Diagnosis ◽  
Sanger Sequencing ◽  
Diagnostic Yield ◽  
Motor Neuropathy ◽  
Peripheral Neuropathies ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Generation Sequencing

PurposeInherited peripheral neuropathies (IPN) represent a large heterogenous group of hereditary diseases with more than 100 causative genes reported to date. In this context, targeted next-generation sequencing (NGS) offers the opportunity to screen all these genes with high efficiency in order to unravel the genetic basis of the disease. Here, we compare the diagnostic yield of targeted NGS with our previous gene by gene Sanger sequencing strategy. We also describe several novel likely pathogenic variants.Design and participantsWe have completed the targeted NGS of 81 IPN genes in a cohort of 123 unrelated patients affected with diverse forms of IPNs, mostly Charcot-Marie-Tooth disease (CMT): 23% CMT1, 52% CMT2, 9% distal hereditary motor neuropathy, 7% hereditary sensory and autonomic neuropathy and 6.5% intermediate CMT.ResultsWe have solved the molecular diagnosis in 49 of 123 patients (~40%). Among the identified variants, 26 variants were already reported in the literature. In our cohort, the most frequently mutated genes are respectively:MFN2,SH3TC2,GDAP1,NEFL,GAN,KIF5AandAARS. Panel-based NGS was more efficient in familial cases than in sporadic cases (diagnostic yield 49%vs19%, respectively). NGS-based search for copy number variations, allowed the identification of three duplications in three patients and raised the diagnostic yield to 41%. This yield is two times higher than the one obtained previously by gene Sanger sequencing screening. The impact of panel-based NGS screening is particularly important for demyelinating CMT (CMT1) subtypes, for which the success rate reached 87% (36% only for axonal CMT2).ConclusionNGS allowed to identify causal mutations in a shorter and cost-effective time. Actually, targeted NGS is a well-suited strategy for efficient molecular diagnosis of IPNs. However, NGS leads to the identification of numerous variants of unknown significance, which interpretation requires interdisciplinary collaborations between molecular geneticists, clinicians and (neuro)pathologists.

Targeted next-generation sequencing using a multigene panel in myeloid neoplasms: Implementation in clinical diagnostics

2017 ◽  
Vol 39 (6) ◽  
pp. 604-612 ◽  
Author(s):  
B. Maes ◽  
J. Willemse ◽  
A. Broekmans ◽  
R. Smets ◽  
B. Cruys ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Clinical Diagnostics ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Myeloid Neoplasms ◽  
Generation Sequencing ◽  
Multigene Panel

Identifying large indels in targeted next generation sequencing assays for myeloid neoplasms: a cautionary tale of the ZRSR1 pseudogene

2017 ◽  
Vol 70 (12) ◽  
pp. 1069-1073 ◽  
Author(s):  
Isaac KS Ng ◽  
Christopher Ng ◽  
Jia Jin Low ◽  
Lily Chiu ◽  
Elaine Seah ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Clinical Diagnostics ◽  
Great Promise ◽  
Next Generation ◽  
Bioinformatics Pipeline ◽  
Targeted Next Generation Sequencing ◽  
Molecular Features ◽  
Myeloid Neoplasms ◽  
Large Indels ◽  
Generation Sequencing

Targeted next generation sequencing platforms have been increasingly utilised for identification of novel mutations in myeloid neoplasms, such as acute myeloid leukaemia (AML), and hold great promise for use in routine clinical diagnostics. In this study, we evaluated the utility of an open source variant caller in detecting large indels in a targeted sequencing of AML samples. While we found that this bioinformatics pipeline has the potential to accurately capture large indels (>20 bp) in patient samples, we highlighted the pitfall of a confounding ZRSR1 pseudogene that led to an erroneous ZRSR2 variant call. We further discuss possible clinical implications of the ZRSR1 pseudogene in myeloid neoplasms based on its molecular features. Knowledge of the confounding ZRSR1 pseudogene in ZRSR2 sequencing assays could be particularly important in AML diagnostics because the detection of ZRSR2 in AML patients is highly specific for an s-AML diagnosis.

scholarly journals Performance evaluation of Sanger sequencing for the diagnosis of primary hyperoxaluria and comparison with targeted next generation sequencing

2014 ◽  
Vol 3 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Emma L. Williams ◽  
Eleanor A. L. Bagg ◽  
Michael Mueller ◽  
Jana Vandrovcova ◽  
Timothy J. Aitman ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Primary Hyperoxaluria ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

Targeted next-generation sequencing panels for monogenetic disorders in clinical diagnostics: the opportunities and challenges

2014 ◽  
Vol 15 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Tom J de Koning ◽  
Jan DH Jongbloed ◽  
Birgit Sikkema-Raddatz ◽  
Richard J Sinke
Keyword(s):  
Next Generation Sequencing ◽  
Clinical Diagnostics ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

Molecular diagnosis of unexplained haemolytic anaemia using targeted next-generation sequencing panel revealed (p.Ala337Thr) novel mutation in GPI gene in two Indian patients

2018 ◽  
Vol 72 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Prabhakar S Kedar ◽  
Vinod Gupta ◽  
Rashmi Dongerdiye ◽  
Ashish Chiddarwar ◽  
Prashant Warang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Haemolytic Anaemia ◽  
Molecular Diagnosis ◽  
Sanger Sequencing ◽  
Genetic Disorder ◽  
Next Generation ◽  
Gold Standard Method ◽  
Targeted Next Generation Sequencing ◽  
Indian Patients ◽  
Generation Sequencing

Glucose-6-phosphate isomerase (GPI) deficiency is an autosomal recessive genetic disorder causing congenital haemolytic anaemia (CHA). Diagnosis of GPI deficiency by the biochemical method is unpredicted. Molecular diagnosis by identifying genetic mutation is the gold standard method for confirmation of disease, but causative genes involved in CHA are numerous, and identifying a gene-by-gene approach using Sanger sequencing is also cumbersome, expensive and labour intensive. Recently, next-generation targeted sequencing is more useful in the diagnosis of unexplained haemolytic anaemia. We used targeted next-generation sequencing (NGS) clinical panel for diagnosis of unexplained haemolytic anaemia in two Indian patients which were pending for a long time. All possible causes of haemolytic anaemia were found within normal limit. NGS by clinical exome panel revealed homozygous novel missense mutation in exon 12, c.1009G>A (p.Ala337Thr) in both patients. We further confirm by measuring red blood cell GPI activity in the patients and showed deficiency whereas parents were having intermediate activity. c.1009G>A mutation was also confirmed by Sanger sequencing of exon 12 of GPI gene. The structural–functional analysis by bioinformatics software like Swiss PDB, PolyPhen-2 and PyMol suggested that this pathogenic variant has a direct impact on the structural rearrangement at the region near the active site of the enzyme. This rapid and high-performance targeted NGS assay can be configured to detect specific CHA mutations unique to an individual defect, making it a potentially valuable method for diagnosis of unexplained haemolytic anaemia.

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