Quality assurance and guidelines for validation of next - IRDiRC

Quality assurance and guidelines for validation of next-generation sequencing tools Gert Matthijs Center for Human Genetics University of Leuven, Belgium - on behalf of EuroGentest -

Genetic testing Molecular testing 1 disease 1 (single) mutation 1 disease 1 gene

different diseases 1 (single) gene

1 disease few genes 1 disease 1 chromosomal anomaly (un)known defect karyotyping

different diseases many genes different diseases different anomalies

total genome

Molecular cytogenetic testing

Cytogenetics G. Matthijs 2011

Long QT syndrome ?

LQT1 LQT2 LQT3 LQT4 LQT5 LQT6 LQT7 LQT8 LQT9 LQT10 LQT11 LQT12 LQT13

ATTGTACGTGATGACCAGTGGAAT ACCGTAAGGTAAAGTACCGTGTAC TTGGTTGGAACGTAGACTGAATGC CAACCCTGGTATTGGTGTCCCGTG TACAAGGTTAGTAATGTACCATTGA TTCCGTAATACGTGTGGCGCGTGC GTAACACACTGACTGACCATCCTG GTAGCTAGTCAAGTCGTAGCTGTC GTGACGTAACGTATATGACACACG TCAGTACGGTCAGTACACACATGC TGTGGTGCAGTACAGATACAGTAC AGATTAGCAGAAATGCAGATTTAGT ATTGTACGTGATGACCAGTGGAAT ACCGTAAGGTAAAGTACCGTGTAC TTGGTTGGAACGTAGACTGAATGC CAACCCTGGTATTGGTGTCCCGTG TACAAGGTTAGTAATGTACCATTGA TTCCGTAATACGTGTGGCGCGTGC GTAACACACTGACTGACCATCCTG GTAGCTAGTCAAGTCGTAGCTGTC GTGACGTAACGTATATGACACACG TCAGTACGGTCAGTACACACATGC TGTGGTGCAGTACAGATACAGTAC AGATTAGCAGAAATGCAGATTTAGT

ATTGTACGTGATGACCAGTGGAAT ACCGTAAGGTAAAGTACCGTGTAC TTGGTTGGAACGTAGACTGAATGC CAACCCTGGTATTGGTGTCCCGTG TACAAGGTTAGTAATGTACCATTGA TTCCGTAATACGTGTGGCGCGTGC GTAACACACTGACTGACCATCCTG

GTAGCTAGTCAAGTCGTAGCTGTC GTGACGTAACGTATATGACACACG TCAGTACGGTCAGTACACACATGC TGTGGTGCAGTACAGATACAGTAC AGATTAGCAGAAATGCAGATTTAGT

K. Devriendt 2012

Next Generation Sequencing (NGS) work flow Targeted sequencing Exome Capture array Library Preparation

Sequencing

Variant Annotation

Courtesy: Erika Souche 2013

Sequence Capture

Demultiplexing

Variant Filtering

Amplification

Mapping

Report

Variant Detection

Illumina run quality control Cluster density

Courtesy: Erika Souche 2013

Sample quality control Base quality score by position

Courtesy: Erika Souche 2013

Mapping

Courtesy: Erika Souche 2013

Diagnostics quality control

Courtesy: Erika Souche 2013

Nat Biotechnology 20 (2012): 1033-36

Nex-StoCT (standardization of clinical testing)

Nat Biotechnology 20 (2012): 1033-36

Nat Biotechnology 20 (2012): 1033-36

Research

≠ Diagnostics

EuroGentest wants to build on these available documents and the work of others, and then comment or amend where necessary.

* US: Nex-StoCT (CDC) Nat Biotechnology 20 (2012): 1033-36 * UK: Targeted NGS Guidelines (CMGS)

* AUSTRALIA: Concept NGS Guidelines (MPS-WG) * THE NETHERLANDS: Concept NGS Guidelines (VKGL) * ESHG PPPC: Recommendations for WGS Draft - Discussed at EuroGentest expert meeting, February 2013

ISSUES of analytical VALIDATION Cfr. US Guidelines

Platform verification: verify the performance specifications established by the manufacturer (e.g., accuracy, precision, etc. (FDA) Test & Bioinformatics pipeline validation: validate system performance characteristics (accuracy, precision, reportable range, reference range, analytical sensitivity, analytical specificity, and other performance characteristics, as applicable) for laboratory-developed tests (LDTs) (CLIA) Reportable range: areas of the targeted region that cannot be sequenced reliably and therefore are excluded from the reportable range Draft - Discussed at EuroGentest expert meeting, February 2013

ISSUES of DEVELOPMENT of the assays Cfr. Dutch Guidelines

* Definition of diagnostic test

* Aim of NGS

* Analytical routing * Core gene list * Diagnostic yield

Draft - Discussed at EuroGentest expert meeting, February 2013

DEFINITION OF A GENETIC TEST

Published evidence for a disease gene to be included in NGS diagnostics (gene list)

This reference has to be included in the labs bibliography Disease genes and mutations that are not amenable to NGS testing (trinucleotide repeat expansions, deletions, …) should not be tested by NGS only (include that information to clinician)

Draft - Discussed at EuroGentest expert meeting, February 2013

AIM OF NGS

- Is the test used to exclude a disease? - Is the test used to confirm a disease?

Depending on this, the approach may differ, and also this will affect how the results will be reported Reporting of ‘uncertain variants’(UVs): no difference with the way UVs will be reported for NGS as compared to current practice

Draft - Discussed at EuroGentest expert meeting, February 2013

CORE GENE LIST

A ‘core gene’ is a disease gene that has to be genotyped completely because it contributes largely/importantly to the disease prevalence. * Other criteria may be: actionable, treatable… * This may include gene dosage analysis

Core genes have to be outlined in the test description Core gene should be outlined in BPG and in CUGC Note: invite experts to generate those (minimal) lists There is an economical aspect in these considerations Draft - Discussed at EuroGentest expert meeting, February 2013

REPORTING, REPORT TO THE CLINICIAN Cfr. Australian guidelines Technical details Quality issues Interpretation (strong preference to only report the known or likely pathogenic variants)

* Requirements: - figure showing the coverage - list of genes that were analysed * Re-contacting / Re-analysis

Draft - Discussed at EuroGentest expert meeting, February 2013

DISTINCTION AND BORDERS BETWEEN RESEARCH AND DIAGNOSTICS Diagnostics is (published) evidence driven Diagnostic test for genes when there is a known relation between genotype and pathology Research is hypothesis driven Any further analysis should be subject to Ethical Board Review Question: where does evidence end? The professional has to decide what is evidence and what is not proven. Draft - Discussed at EuroGentest expert meeting, February 2013