20.02.17 NGS- service delivery and patient pathways (panel design)

Question 1

Different types of target enrichment methods

Answer 1

• PCR amplicon-based strategies (Raindance). Not scalable and could suffer allele drop out due to rare polymorphisms
• Hybrid capture (Agilent SureSelect). Difficulty distinguishing between genes and their pseudogenes
• Haloplex (e.g. Agilent). Amplicon based and obviates the need for library construction

Question 2

Considerations when choosing genes to be in panels

Answer 2

• Published evidence for gene involvement in disease
• Core gene list to enable uniformity of testing between labs (Genomics England PanelApp)
• Smaller gene panels to maintain high read coverage. Important for confident calling of mosaics of low level heterogeneity.

Question 3

What is PanelApp (Genomics England)

Answer 3

Crowdsourcing tool to allow virtual gene panels to be shared and evaluated in scientific community.

Question 4

Why use PanelApp

Answer 4

• Encourages standardisation of gene panels based on expert knowledge and guidelines.
• Allows evidence for research grade genes to be collected. Could lead to them being promoted to clinical grade as more supportive evidence emerges.

Question 5

Considerations for gene transcripts

Answer 5

-Validation document should include justification for selecting a transcript. If there are two with unique exons, then both should be included.

Question 6

What is LRG project

Answer 6

• Locus Reference Genomic project
• Aims to create universally accepted reference standards for variant reporting.
• Each LRG has a fixed section (stable genomic DNA sequence for region of interest) and updatable section (most recent biological information)

Question 7

Why are DNA quality checks needed prior to sequencing

Answer 7

• NGS runs are costly, want to check quality is adequate before starting.
• Some DNA is irreplaceable (deceased, TOPs) and needs to be of sufficient quality.

Question 8

Why are samples barcoded

Answer 8

To allow multiplexing of samples, decreases cost per patient.

Question 9

Why is validation an important part of service delivery

Answer 9

• Looks at reproducibility and robustness.
• Identifies technical weakness
• To determine the sensitivity and error rate.

Question 10

What quality control schemes should be done

Answer 10

• IQC (internal quality control). Done for each run. Monitor raw data to ensure it is not suboptimal.
• EQA (external quality assessment). External DNAs of known genotype are distributed to labs for testing using their routine procedure. Also analytical exercises (supplied FASTQ files).

Question 11

What do NGS best practice guidelines say about confirmation of variants

Answer 11

An independent test on a new dilution should be done if there is no robust tube transfer checking system (barcode scanning/ witnessed transfers) or no secondary test (SNP assay) to assure sample identity.

Question 12

How should reports be formatted

Answer 12

• Scientific section: Follow ACGS BP guidelines. Vairants annotated using HGVS recommendations. Include clinical information provided in referral. Negative results should have expected diagnostic yield if known.
• Technical section: Information about panel (genes tested, transcript number, method), coverage (also areas lacking coverage), VUSs, incidental findings.

Question 13

Considerations for pricing

Answer 13

• Costs are decreasing but some things are fixed, analysis time, reagents
• Batching allows more samples per run, reducing cost (although will decrease read depth, limited by library prep method and sequencing platform)
• Cost needs to be competitive (NGS usually cheaper than sequential Sanger analysis)

Question 14

What is the translational benefits of NGS in patient pathway

Answer 14

• Gives us an understanding of human variation and association with disease risk
• Can be used with an individuals response to treatment
• Aids clinical decision making

Question 15

Challenges of NGS in patient pathways

Answer 15

• Interpretation of results (VUSs, incidental findings, reduced penetrant alleles)
• Ethical issues for probands and relatives
• Services may not be able to cope with testing outcomes in terms of counselling and medical follow up (reanalysis of data, VUS re-interpretation)

Question 16

What does genetic counselling model consist of

Answer 16

1. Pre-test counselling:
   - Discussion on range of outcomes of testing (including unanticipated results, VUSs)
   - If panel testing, then they must be educated on the implications of testing all genes.
   - Whether the results should be held locally or on their medical record (insurance-based discrimination)
2. Post-test counselling
   - Inform patients of updates on mutations identified and whether new treatments available for them.