Standardisation of sequencing coverage depth in NGS: recommendation for detection of clonal and subclonal mutations in cancer diagnostics

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00098892%3A_____%2F19%3AN0000032" target="_blank" >RIV/00098892:_____/19:N0000032 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/61989592:15110/19:73594809

Výsledek na webu

<a href="https://www.frontiersin.org/articles/10.3389/fonc.2019.00851/full" target="_blank" >https://www.frontiersin.org/articles/10.3389/fonc.2019.00851/full</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3389/fonc.2019.00851" target="_blank" >10.3389/fonc.2019.00851</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Standardisation of sequencing coverage depth in NGS: recommendation for detection of clonal and subclonal mutations in cancer diagnostics

Popis výsledku v původním jazyce

The insufficient standardization of diagnostic next-generation sequencing (NGS) still limits its implementation in clinical practice, with the correct detection of mutations at low variant allele frequencies (VAF) facing particular challenges. We address here the standardization of sequencing coverage depth in order to minimize the probability of false positive and false negative results, the latter being underestimated in clinical NGS. There is currently no consensus on the minimum coverage depth, and so each laboratory has to set its own parameters. To assist laboratories with the determination of the minimum coverage parameters, we provide here a user-friendly coverage calculator. Using the sequencing error only, we recommend a minimum depth of coverage of 1,650 together with a threshold of at least 30mutated reads for a targeted NGSmutation analysis of≥3% VAF, based on the binomial probability distribution. Moreover, our calculator also allows adding assay-specific errors occurring during DNA processing and library preparation, thus calculating with an overall error of a specific NGS assay. The estimation of correct coverage depth is recommended as a starting point when assessing thresholds of NGS assay. Our study also points to the need for guidance regarding the minimum technical requirements, which based on our experience should include the limit of detection (LOD), overall NGS assay error, input, source and quality of DNA, coverage depth, number of variant supporting reads, and total number of target reads covering variant region. Further studies are needed to define the minimum technical requirements and its reporting in diagnostic NGS.

Název v anglickém jazyce

Standardisation of sequencing coverage depth in NGS: recommendation for detection of clonal and subclonal mutations in cancer diagnostics

Popis výsledku anglicky

The insufficient standardization of diagnostic next-generation sequencing (NGS) still limits its implementation in clinical practice, with the correct detection of mutations at low variant allele frequencies (VAF) facing particular challenges. We address here the standardization of sequencing coverage depth in order to minimize the probability of false positive and false negative results, the latter being underestimated in clinical NGS. There is currently no consensus on the minimum coverage depth, and so each laboratory has to set its own parameters. To assist laboratories with the determination of the minimum coverage parameters, we provide here a user-friendly coverage calculator. Using the sequencing error only, we recommend a minimum depth of coverage of 1,650 together with a threshold of at least 30mutated reads for a targeted NGSmutation analysis of≥3% VAF, based on the binomial probability distribution. Moreover, our calculator also allows adding assay-specific errors occurring during DNA processing and library preparation, thus calculating with an overall error of a specific NGS assay. The estimation of correct coverage depth is recommended as a starting point when assessing thresholds of NGS assay. Our study also points to the need for guidance regarding the minimum technical requirements, which based on our experience should include the limit of detection (LOD), overall NGS assay error, input, source and quality of DNA, coverage depth, number of variant supporting reads, and total number of target reads covering variant region. Further studies are needed to define the minimum technical requirements and its reporting in diagnostic NGS.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

30205 - Hematology

Projekt

<a href="/cs/project/NV16-32339A" target="_blank" >NV16-32339A: Vliv funkčních polymorfismů ovlivňujících zánět a oxidační stres na průběh chronické lymfocytární leukémie a volbu individuální léčebné strategie</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Frontiers in Oncology

ISSN

2234-943X

e-ISSN

2234-943X

Svazek periodika

9

Číslo periodika v rámci svazku

September 2019

Stát vydavatele periodika

CH - Švýcarská konfederace

Počet stran výsledku

6

Strana od-do

851

Kód UT WoS článku

000483735300001

EID výsledku v databázi Scopus

2-s2.0-85073060022