scholarly journals An adaptive method of defining negative mutation status for multi-sample comparison using next-generation sequencing

2021 ◽  
Vol 14 (S2) ◽  
Author(s):  
Nicholas Hutson ◽  
Fenglin Zhan ◽  
James Graham ◽  
Mitsuko Murakami ◽  
Han Zhang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Tumor Cell ◽  
Null Hypothesis ◽  
False Negative ◽  
Adaptive Method ◽  
Positive Sample ◽  
Next Generation ◽  
Mutation Status ◽  
Cell Fractions ◽  
Generation Sequencing

Abstract Background Multi-sample comparison is commonly used in cancer genomics studies. By using next-generation sequencing (NGS), a mutation's status in a specific sample can be measured by the number of reads supporting mutant or wildtype alleles. When no mutant reads are detected, it could represent either a true negative mutation status or a false negative due to an insufficient number of reads, so-called "coverage". To minimize the chance of false-negative, we should consider the mutation status as "unknown" instead of "negative" when the coverage is inadequately low. There is no established method for determining the coverage threshold between negative and unknown statuses. A common solution is to apply a universal minimum coverage (UMC). However, this method relies on an arbitrarily chosen threshold, and it does not take into account the mutations' relative abundances, which can vary dramatically by the type of mutations. The result could be misclassification between negative and unknown statuses. Methods We propose an adaptive mutation-specific negative (MSN) method to improve the discrimination between negative and unknown mutation statuses. For a specific mutation, a non-positive sample is compared with every known positive sample to test the null hypothesis that they may contain the same frequency of mutant reads. The non-positive sample can only be claimed as “negative” when this null hypothesis is rejected with all known positive samples; otherwise, the status would be “unknown”. Results We first compared the performance of MSN and UMC methods in a simulated dataset containing varying tumor cell fractions. Only the MSN methods appropriately assigned negative statuses for samples with both high- and low-tumor cell fractions. When evaluated on a real dual-platform single-cell sequencing dataset, the MSN method not only provided more accurate assessments of negative statuses but also yielded three times more available data after excluding the “unknown” statuses, compared with the UMC method. Conclusions We developed a new adaptive method for distinguishing unknown from negative statuses in multi-sample comparison NGS data. The method can provide more accurate negative statuses than the conventional UMC method and generate a remarkably higher amount of available data by reducing unnecessary “unknown” calls.

scholarly journals Frequency detection of BRAF V600E mutation in a cohort of pediatric langerhans cell histiocytosis patients by next-generation sequencing

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Shunqiao Feng ◽  
Lin Han ◽  
Mei Yue ◽  
Dixiao Zhong ◽  
Jing Cao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Langerhans Cell Histiocytosis ◽  
Mutation Screening ◽  
Langerhans Cell ◽  
Braf V600e ◽  
P Value ◽  
Type I ◽  
Next Generation ◽  
Mutation Status ◽  
Generation Sequencing

Abstract Background Langerhans cell histiocytosis (LCH) is a rare neoplastic disease that occurs in both children and adults, and BRAF V600E is detected in up to 64% of the patients. Several studies have discussed the associations between BRAF V600E mutation and clinicopathological manifestations, but no clear conclusions have been drawn regarding the clinical significance of the mutation in pediatric patients. Results We retrieved the clinical information for 148 pediatric LCH patients and investigated the BRAF V600E mutation using next-generation sequencing alone or with droplet digital PCR. The overall positive rate of BRAF V600E was 60/148 (41%). The type of sample (peripheral blood and formalin-fixed paraffin-embedded tissue) used for testing was significantly associated with the BRAF V600E mutation status (p-value = 0.000 and 0.000). The risk of recurrence declined in patients who received targeted therapy (p-value = 0.006; hazard ratio 0.164, 95%CI: 0.046 to 0.583). However, no correlation was found between the BRAF V600E status and gender, age, stage, specific organ affected, TP53 mutation status, masses close to the lesion or recurrence. Conclusions This is the largest pediatric LCH study conducted with a Chinese population to date. BRAF V600E in LCH may occur less in East Asian populations than in other ethnic groups, regardless of age. Biopsy tissue is a more sensitive sample for BRAF mutation screening because not all of circulating DNA is tumoral. Approaches with low limit of detection or high sensitivity are recommended for mutation screening to avoid type I and II errors.

Effective Assessment of egfr Mutation Status in Bronchoalveolar Lavage and Pleural Fluids by Next-Generation Sequencing

2012 ◽  
Vol 19 (3) ◽  
pp. 691-698 ◽  
Author(s):  
Fiamma Buttitta ◽  
Lara Felicioni ◽  
Maela Del Grammastro ◽  
Giampaolo Filice ◽  
Alessia Di Lorito ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Bronchoalveolar Lavage ◽  
Egfr Mutation ◽  
Next Generation ◽  
Mutation Status ◽  
Effective Assessment ◽  
Generation Sequencing

Somatic POLE proofreading domain mutations by next generation sequencing to predict outcomes in stage II colorectal cancer.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 559-559
Author(s):  
Shaobo Mo ◽  
Yaqi Li ◽  
Junjie Peng
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Instability ◽  
Disease Free Survival ◽  
Stage Ii ◽  
Molecular Heterogeneity ◽  
Next Generation ◽  
Wild Type ◽  
Mutation Status ◽  
Pathologic Features ◽  
Generation Sequencing

559 Background: Stage II colorectal cancers (CRC) exhibit unique molecular heterogeneity and patients with somatic POLE mutations are defined as a distinct tumor subgroup. The aim of this study was to clarify the characteristics and prognostic effect of somatic POLE proofreading domain mutations by Next Generation Sequencing in stage II CRC. Methods: Recurrent patients were 1:1 matched to patient with no recurrence for stage II CRC patients diagnosed between 2008 and 2013 in Fudan University Shanghai Cancer Center (FUSCC). All patients were pathologically confirmed and received radical resection. Microsatellite instability status and POLE mutation status were determined by NGS using ColonCore panel (Burning Rock, Guangzhou, China). Groups based on NGS-POLE mutation status were compared in terms of patient demographics and pathologic features using chi-squared tests. Survival curves were plotted using the Kaplan-Meier method. Results: In total, 245 stage II CRC patients were enrolled. POLE mutations were detected in 25 (10.2%) of 245 stage II CRC, while only 9 (3.7%) was located in proofreading domain (NGS-POLE EDM-mutations) and other 16 were found in non-proofreading domain (non-NGS-POLE mutations). Compared to cases with wild-type POLE, cases with NGS-POLE EDM-mutations were more prone to be microsatellite instability-high (MSI-H) (3 of 9 [33.3%] vs. 23 of 236 [9.7%], p = 0.024), younger at diagnosis (median 46 years vs. 62 years, p < 0.001), and more frequently with right-sided tumor location (6 of 9 [66.7%] vs. 54 of 236 [22.9%]; p = 0.003). All tumors with NGS-POLE EDM-mutations presented with a surprisingly high tumor mutation burden (TMB) (median 145.2 per Mb). Patients with NGS-POLE EDM-mutations have favorable 5-year disease-free survival (DFS) compared with those with non-NGS-POLE EDM-mutations and wild-type POLE (100% vs. 62.5% vs. 52.5%, p = 0.037). Though there is DFS difference between patients with non-NGS-POLE EDM-mutations and wild-type POLE, the difference was not statistically significant (p = 0.412). Conclusions: Stage II CRC patients with NGS-POLE EDM-mutation identify a special cancer subset with better immune environment predicting excellent outcomes.

scholarly journals Hidden biases in germline structural variant detection

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Michael M. Khayat ◽  
Sayed Mohammad Ebrahim Sahraeian ◽  
Samantha Zarate ◽  
Andrew Carroll ◽  
Huixiao Hong ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
False Negative ◽  
False Negative Rate ◽  
Next Generation Sequencing Data ◽  
Chinese Family ◽  
Next Generation ◽  
Sequencing Data ◽  
Structural Variations ◽  
The Impact ◽  
Generation Sequencing

Abstract Background Genomic structural variations (SV) are important determinants of genotypic and phenotypic changes in many organisms. However, the detection of SV from next-generation sequencing data remains challenging. Results In this study, DNA from a Chinese family quartet is sequenced at three different sequencing centers in triplicate. A total of 288 derivative data sets are generated utilizing different analysis pipelines and compared to identify sources of analytical variability. Mapping methods provide the major contribution to variability, followed by sequencing centers and replicates. Interestingly, SV supported by only one center or replicate often represent true positives with 47.02% and 45.44% overlapping the long-read SV call set, respectively. This is consistent with an overall higher false negative rate for SV calling in centers and replicates compared to mappers (15.72%). Finally, we observe that the SV calling variability also persists in a genotyping approach, indicating the impact of the underlying sequencing and preparation approaches. Conclusions This study provides the first detailed insights into the sources of variability in SV identification from next-generation sequencing and highlights remaining challenges in SV calling for large cohorts. We further give recommendations on how to reduce SV calling variability and the choice of alignment methodology.

scholarly journals 667. Next Generation Sequencing of Microbial Cell Free DNA in the Diagnosis and Treatment of Infectious Disease in Children: When Does the Result Justify the Cost?

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S436-S436
Author(s):  
Rachel Downey Quick ◽  
Kelli A Martinez ◽  
Susan M Russo ◽  
Sarah E McGwier ◽  
Rachel A Quirt ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Antimicrobial Resistance ◽  
False Negative ◽  
Next Generation ◽  
Cell Free Dna ◽  
Negative Results ◽  
Free Dna ◽  
False Negative Results ◽  
The Cost ◽  
Generation Sequencing

Abstract Background Pathogen testing using next-generation sequencing of microbial cell-free DNA (NGS cfDNA) is a promising diagnostic tool to identify pathogens that might not be detected using conventional lab evaluation. Considering the cost of this test, it is important to determine when it is most useful to the plan of care (POC). Figure 1. Unit of admission among cases Figure 2. Patient characteristics in cases determined to be valuable and not valuable to the plan of care (POC) Methods In this retrospective study, we collected data from the medical charts of 50 consecutive NGS cfDNA tests in a free-standing children’s hospital. We evaluated patients for demographics, underlying conditions, diagnosis at time of testing, conventional laboratory testing and timing, medical treatment, and NGS cfDNA test results for clinical relevance or false negative results compared to conventional testing. The primary goal was to identify patients for whom the NGS cfDNA testing affected the POC. Charts were reviewed, and determinations regarding whether the result influenced the POC were confirmed by a provider. Results We were unable to differentiate patients with clinically valuable NGS cfDNA results (Fig 1 & 2). Among those with NGS cfDNA results valuable to the POC (n=22), both negative and positive testing guided POC (13 valuable negative vs. 9 diagnostic cases). In the total sample, 5 cases (10%) had a clinically relevant pathogen identified through conventional testing, but not through NGS cfDNA and 2 cases had antimicrobial resistance on culture, which is not detected by NGS cfDNA. Conclusion While we did not find a specific clinical profile for NGS cfDNA use, positive results were essential to the diagnosis in 18% of cases with otherwise negative laboratory evaluation for the pathogen identified in NGS cfDNA. Negative tests affected the POC in 26% of cases by avoiding unnecessary antimicrobials in high risk immunocompromised patients and patients that presented with low-risk of infection, but unclear disease process. Caution must be exercised with reliance on this test with respect to antimicrobial resistance and risk of false negative results. Disclosures All Authors: No reported disclosures

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

2018 ◽  
Vol 143 (4) ◽  
pp. 463-471 ◽  
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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