18.02.17 NGS service delivery

Question 1

What are the consideration when selecting genes to be included in a NGS panel?

Answer 1

1) Selection of genes in collaboratorion with clinicains for which there is published evidence of disease involvements
2) DIagnostic yield from a NGS panel should at least equal that of sequential Sanger gene sequencing
3) Including genes with tenuous evidence of disease involvement will decrease diagnostic yuled (clinical sensitivity of a test) and lead to greater VUS.
4) LImiting gene number will maintain high coverage of targets increasing confidence of calls in mosaics/ low level heterogentity (e.g. for oncology or mt applications).
5) Commerical gene panels available for some applications
6) Moving to virtual panels may mean gene selection is offered on the presence of specific phenotype/clinical features.
7) Panel App/test directory.

Question 2

What are benefits of Genomics England PanelApp?

Answer 2

1) Crowdsourcing tool to allow gene panels to be shared, downloaded, viewed and evaluated by scientific community
2) Encourages standardisation of gene panels based on expert knowledge and guidelines
3) The PanelApp also allows evidence for research grade genes to be collected, which may eventually be promoted to clinical grade as more evidence emerges

Question 3

What is the significance of pseudogenes in NGS?

Answer 3

Presence of pseudogenes may sequester some baits leading to poorer tiling across some regions. However, during the mapping process of reads, more than one alignment usually result in both being discarded by mapping software - may need to Sanger fill

Question 4

Why are DNA quality checks essential prior to NGS?

Answer 4

NGS run is expensive.
Re-precipitate is required
Precious samples e.g. from deceased patients is irreplaceable.

Question 5

What is the benefit of barcoding samples?

Answer 5

Allows pooling of samples which decreases cost per patient.

Question 6

What are virtual panels?

Answer 6

Where genes span overlapping phenotypes, allow sub-panel analysis bioinformatically (analysis of a sub-set of genes). eg. for hypertrophic cardiomyopathy specifically when a cardiomyopathy panel is used.

With more widespread use of clinical exomes within diagnostic labs, services may move towards automatic virtual panel selection based on specific phenotypes/clinical features provided at time of request

Question 7

What guidelines are in place for NGS sequencing and analysis?

Answer 7

ACGS Practise guidelines for targeted NGS sequencing and analysis were approved and released in 2014 recognise that different centres are using different platforms, library preparation methods and analysis pipelines, but ‘aim to establish consensus standards for identifying and reporting mutations’. sections on validation, quality, data analysis, data storage, and reporting.

Question 8

What steps are required in NGS validation?

Answer 8

Required on all aspects of the test process: targeting methods, sequencing processing and data analysis.

Important to understand the technical weaknesses
ensure that these are safely assessed during validation,
e.g. Homopolymer tract errors that vary according to platform and lower sensitivity for detecting base substitutions compared to insertions and deletions.

the sequencing platform (instrument) should have undergone validation by combining performance data across all tests developed by a laboratory: maximizing the number and types of variants tested across a broad range of genomic regions can tighten confidence intervals

Reproducibility and robustness need to be assessed (particularly in terms of horizontal coverage).: BPG recommend validation samples are analysed from at least 3 independent runs
run-to-run comparisons will help determine the level of multiplexing possible to ensure minimum diagnostic coverage.

It is acceptable to perform validation at the level of the process rather than the individual gene.

Positive control samples should be representative of the disease mutation spectrum

UKGTN requires that new panels and addition of genes to existing panels should be validated using a ‘known normal control’ from the 1000 genomes project.

Question 9

What are the sensitivity considerations when designing a NGS assay?

Answer 9

Sensitivity

the required sensitivity will depend upon clinical application.

BPG state it is essential for labs to demonstrate 95% confidence that the error rate for heterozygote/homozygote mutation detection is <5% (recommended <2%) (see Mattocks et al 2010).

this requires concordant results for a minimum of 60 unique variants (recommended 150) tested by the new method in an independent, blinded analysis

Question 10

What are the IQC/EQA considerations for NGS assays?

Answer 10

IQC should be undertaken for each run.

1. various QC metrics produced from each run, from the ‘sequencer’ and also for the raw data and the filtered data, such as cluster density, number of reads, coverage etc.
2. Robust checking and recording of these metrics should be in place, to ensure that sub-optimal data is not reported.
3. Some of this technical data should be included in the technical report - see below.

EQA

1. UKNEQAS continue to offer pilot schemes for NGS for both somatic and germ line testing ran a pilot schemes
2. wet based exercise where one genomic DNA sample is distributed for testing using the laboratory’s routine NGS procedure (single gene, panel test or exome sequencing)
3. a dry based exercise involving the analysis of data (supplied as FASTQ files.)

Question 11

What is the process of NGS variant confirmation?

Answer 11

The NGS practice guidelines state:

in the absence of a proven, robust tube transfer checking system (e.g. barcode scanning, witnessed transfers) or a secondary test (e.g. SNP assay) to assure sample identify at each stage, it is essential to confirm mutations or variants included in a clinical report by independent test from a new DNA dilution.

This may involve designing a large number of news primers for genes not previously tested,

but does ensure that the primers are available if cascade testing for the variant is undertaken for family members in the future.

Confirmation of variants/mutations may be by Sanger sequencing, MLPA or other techniques, depending on the variant

The testing will also provide additional data regarding test sensitivity (false positive rate) to support discontinuation of confirmatory testing once secondary identity testing has been validated.

Question 12

What should be included in the scientific component of a NGS report?

Answer 12

should follow standard ACGS BPG for report writing

should integrate sequence data with clinical information that has been provided.

Variants should be annotated according to HGVS recommendations.

Negative reports should include the expected diagnostic yield if known (i.e. the proportion of cases with a phenotype in question in which a mutation has been detected by the testing strategy employed).

Question 13

What should be included in the technical component of a NGS report?

Answer 13

all supportive information about the panel, including genes tested (with reference sequence ID +/- OMIM number), plus information about splice site and promoter regions included

the methods used (library preparation methods, analyser equipment used and overview of bioinformatics pipelines and software)

coverage achieved and highlight any regions that may not have been adequately covered

list any unique variant of unknown clinical significance.

whether dosage has been robustly analysed.

Question 14

What are the price considerations when designing a NGS assay?

Answer 14

Reagent costs
Analysis time
Batching and barcoding allows more patients but is limited by the library prep and platform and increasing the number of patients will decrease read depth.
Cost needs to be competitive (NGS usually cheaper than sequential Sanger for heterogenous disorders)

Question 15

What is the translational utility of NGS?

Answer 15

understanding the human genetic variation and its association with disease risk

individual response to treatment

interpretation and translation of data for clinical decision making.

With appealing characteristics as high test accuracy, number of conditions tested and rapid turn-around time, genomic sequencing is “expected to become a central piece of routine healthcare management which can be practiced regularly by physicians from their offices”.

Question 16

What are the challenges of NGS?

Answer 16

clinical utility

interpretation of results

detection of variants of unknown clinical significance and incidental findings

ethical issues for the index case and their relatives

healthcare services might be unable to adequately cope with testing outcomes in terms of counselling and proper medical follow-up

Question 17

What are the considerations when deciding between single gene test and a panel test?

Answer 17

Multiple factors to consider, e.g.:

a disease panel test may lead to an improved detection rate for the causative gene mutations,

depending on the findings there may be not sufficient data in the medical literature to make them clinically actionable

the improved detection rate of such panel should be weighed against a higher risk to find a variant of unknown significance.

every genome sequenced is heterozygous for 50–100 variants classified by the Human Gene Mutation Database (HGMD) as causing inherited disorders and often has many more functional homozygous or heterozygous novel variants in genes implicated in Mendelian disease.

also higher risk of an incidental finding

Genotyping errors

Annotations incorrect on HGMD

Variable penetrance of mutations

Question 18

What are the genetic counselling considerations for NGS?

Answer 18

Any patient giving consent to the use of NGS must be carefully counselled about the potential of the technology to reveal information that may be relevant to their risk of future disease, and understand how this information will be handled.

Question 19

What are the two stages of genetic counselling for NGS?

Answer 19

pre-test genetic counselling

needs to involve a discussion of the range of information that could be learned via an NGS test, as well as the increased risk of discovering unanticipated results and variants of unknown significance.

If a gene panel is being tested by NGS, the patients must be educated about the implication of all genes included.

Post-test genetic counselling

clinicians should continue to inform patients on updates on the mutations found in their testing, new treatment for the mutated genes.

Question 20

How do the American National Society of Genetic Counsellors suggest stratification of genes found on NGS cancer panels?

Answer 20

The American National Society of Genetic Counsellors suggests stratification of genesf ound on NGS cancer panels into 3 categories:

Category 1: Genes that, when mutated, confer high cancer risks, with published management guidelines for those with mutations. E.g. TP53, APC, BRCA1.

Category 2: Genes that, when mutated, confer moderate cancer risks, with no management guidelines for those with mutations. Eg ATM

Category 3: Genes that, when mutated, are known to be prevalent within a certain cancer patient population , however the degree for cancer risk and tumour spectrum are not well understood, with no management guidelines for those with mutations. Eg BRIP1

Question 21

What are the ethical principles of using NGS in human research?

Answer 21

1. Any mutation believed to account for the disease being studied should be communicated to the patient after due validation, and used to guide possible treatment and genetic counselling.
2. For the purposes of the UK10K project, researchers do not have an obligation to seek out incidental findings for, among other considerations, such an obligation would dramatically impede researchers’ ability to focus on addressing the particular disease state being studied.
3. Suggested that where potentially clinically important mutations are identified, the genotype should be validated by an accredited diagnostic laboratory, and the clinical validity (whether the results are of consistent clinical significance) and clinical utility (whether knowledge of the results is likely to benefit the patient) carefully considered in conjunction with a clinical geneticist.

Whatever the particular position adopted by individual research teams, it is clear that the consent gained must be truly informed, with explicit statements incorporated about handling genetic information in these different scenarios.

This approach to using NGS in human research volunteers represents a starting position, arrived at after multidisciplinary deliberations, and it is expected to evolve as experience grows. Generating guidelines for the clinical diagnostic use of NGS is likely to be more complex still.