In Silico Proficiency Testing for Clinical Next-Generation Sequencing

Context.—Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample. Objective.—To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically. Design.—DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. Results.—Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. Conclusions.—The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.

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In-Silico Prediction of Candidate SNPs in TRIOBP, TMC1 and EYA4 Genes Causing Hereditary Deafness in Three Sudanese Patients Using Next Generation Sequencing

Nova Journal of Medical and Biological Sciences ◽

10.20286/nova-jmbs-030235 ◽

2014 ◽

Vol 03 (02) ◽

pp. 1-10

Author(s):

Mahmoud Eltayeb Koko Musa ◽

Yousuf Hasan Yousuf Bakhit ◽

Ashraf Yahia Osman Mohamed ◽

Asaad Tageldein Idris ◽

Ashraf Ahmed Mohamed Ahmed Fadul ◽

...

Keyword(s):

Next Generation Sequencing ◽

In Silico ◽

Candidate Snps ◽

In Silico Prediction ◽

Next Generation ◽

Hereditary Deafness ◽

Generation Sequencing

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Performance Comparison of Different Analytic Methods in Proficiency Testing for Mutations in the BRAF, EGFR, and KRAS Genes: A Study of the College of American Pathologists Molecular Oncology Committee

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2018-0396-cp ◽

2019 ◽

Vol 143 (10) ◽

pp. 1203-1211 ◽

Cited By ~ 2

Author(s):

Joel T. Moncur ◽

Angela N. Bartley ◽

Julia A. Bridge ◽

Suzanne Kamel-Reid ◽

Alexander J. Lazar ◽

...

Keyword(s):

Next Generation Sequencing ◽

Proficiency Testing ◽

Critical Issue ◽

Performance Comparison ◽

Test Method ◽

Next Generation ◽

Molecular Oncology ◽

Assay Methods ◽

Commercial Kits ◽

Generation Sequencing

Context.— The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories (“home brews”), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee. Objective.— To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes: BRAF, EGFR, and KRAS. Design.— There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences for BRAF, EGFR, and KRAS. Performance was analyzed by test method, kit manufacturer, variants tested, and preanalytic and postanalytic practices. Results.— Of 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses for BRAF; 848 [97.5%] acceptable of 870 total responses for EGFR; 942 [97.0%] acceptable of 971 total responses for KRAS). Next-generation sequencing platforms (summed across all analytes and 2 platforms) demonstrated 99.4% accuracy for these analytes (165 [99.4%] acceptable of 166 total next-generation sequencing responses). Slight differences in performance were noted among select commercial assays, dependent upon the particular design and specificity of the assay. Wide differences were noted in the lower limits of neoplastic cellularity laboratories accepted for testing. Conclusions.— These data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.

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From FASTQ to Function: In Silico Methods for Processing Next-Generation Sequencing Data

Methods in Molecular Biology - Clostridium difficile ◽

10.1007/978-1-4939-6361-4_3 ◽

2016 ◽

pp. 23-33 ◽

Cited By ~ 1

Author(s):

Mark D. Preston ◽

Richard A. Stabler

Keyword(s):

Next Generation Sequencing ◽

In Silico ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Generation Sequencing ◽

In Silico Methods

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Proficiency Testing of Standardized Samples Shows Very High Interlaboratory Agreement for Clinical Next-Generation Sequencing–Based Oncology Assays

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2018-0336-cp ◽

2018 ◽

Vol 143 (4) ◽

pp. 463-471 ◽

Cited By ~ 9

Author(s):

Jason D. Merker ◽

Kelly Devereaux ◽

A. John Iafrate ◽

Suzanne Kamel-Reid ◽

Annette S. Kim ◽

...

Keyword(s):

Next Generation Sequencing ◽

Proficiency Testing ◽

False Negative ◽

High Specificity ◽

Variant Allele ◽

Next Generation ◽

Single Nucleotide Variants ◽

Sequencing Platform ◽

Negative Results ◽

Generation Sequencing

Context.— Next-generation sequencing–based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. Objective.— To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. Design.— CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. Results.— The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. Conclusions.— Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types.

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Proficiency Testing of Standardized Samples Shows High Interlaboratory Agreement for Clinical Next-Generation Sequencing–Based Hematologic Malignancy Assays With Survey Material–Specific Differences in Variant Frequencies

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2019-0352-cp ◽

2020 ◽

Vol 144 (8) ◽

pp. 959-966 ◽

Cited By ~ 1

Author(s):

Alissa Keegan ◽

Julia A. Bridge ◽

Neal I. Lindeman ◽

Thomas A. Long ◽

Jason D. Merker ◽

...

Keyword(s):

Next Generation Sequencing ◽

Proficiency Testing ◽

Hematologic Malignancy ◽

False Negative ◽

False Negative Rate ◽

Hematologic Malignancies ◽

Variant Allele ◽

Next Generation ◽

Single Nucleotide Variants ◽

Generation Sequencing

Context.— As laboratories increasingly turn from single-analyte testing in hematologic malignancies to next-generation sequencing–based panel testing, there is a corresponding need for proficiency testing to ensure adequate performance of these next-generation sequencing assays for optimal patient care. Objective.— To report the performance of laboratories on proficiency testing from the first 4 College of American Pathologists Next-Generation Sequencing Hematologic Malignancy surveys. Design.— College of American Pathologists proficiency testing results for 36 different engineered variants and/or allele fractions as well as a sample with no pathogenic variants were analyzed for accuracy and associated assay performance characteristics. Results.— The overall sensitivity observed for all variants was 93.5% (2190 of 2341) with 99.8% specificity (22 800 of 22 840). The false-negative rate was 6.5% (151 of 2341), and the largest single cause of these errors was difficulty in identifying variants in the sequence of CEBPA that is rich in cytosines and guanines. False-positive results (0.18%; 40 of 22 840) were most likely the result of preanalytic or postanalytic errors. Interestingly, the variant allele fractions were almost uniformly lower than the engineered fraction (as measured by digital polymerase chain reaction). Extensive troubleshooting identified a multifactorial cause for the low variant allele fractions, a result of an interaction between the linearized nature of the plasmid and the Illumina TruSeq chemistry. Conclusions.— Laboratories demonstrated an overall accuracy of 99.2% (24 990 of 25 181) with 99.8% specificity and 93.5% sensitivity when examining 36 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 10% or greater. The data also highlight an issue with artificial linearized plasmids as survey material for next-generation sequencing.

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