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Flora Ross > NGS methods and applications > Flashcards

NGS methods and applications Flashcards

1
Q

Purpose of library prep

3 main steps

A

Prepares DNA for sequencing, depending on NGS platform used

Fragmentation
Adaptor/indices ligation
Enrichment

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2
Q

Purpose of an enrichment step

A

Capture the ROI for single genes, targeted panels or clinical/whole exome

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3
Q

2 types of enrichment

A

Amplicon/PCR based

Hybridisation/Capture based

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4
Q

2 advantages of amplicon enrichment

A

Lower cost and faster TAT

Decreased amount of starting DNA

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5
Q

Name an example kit that uses amplicon enrichment and an application

A

QIAseq for somatic cancer panels

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6
Q

3 disadvantages of amplicon enrichment

A

Preferential amplification = non-uniform enrichment

Artefacts

Difficult to multiplex for high ROI numbers

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7
Q

What are UMIs?

A

Unique Molecular Indices are used to tag an original DNA frag. Allow filtering of PCR duplicate reads based on UMI. Can also be used to identify CNVs

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8
Q

Basis of capture enrichment

A

RNA or DNA oligonucleotides specific to target ROI and ‘pulled down’ using biotin and streptavidin labelled bead clean up

Creates a tile of captured fragments that represent a whole ROI

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9
Q

3 advantages of capture based enrichment

A

More uniform coverage and improved for GC rich regions

PCR duplicates are easily recognised and removed

Higher read depth can be achieved for CNV/mosaicism analysis

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10
Q

2 limitations of capture enrichment

A

More starting material required

High costs and slower TAT

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11
Q

1 example kit using capture enrichment

A

Agilent SureSelect

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12
Q

Describe short read NGS

A

Series of automatically coordinated repeated chemical reactions, carried out in a flow cell which has immobilised templates and reagents.

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13
Q

What is the main sequencing method for SRS and outline the process

A

Sequencing by synthesis

Repeated cyclical process involving nucleotide addition, washing and signal detection

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14
Q

Briefly describe Illumina SBS sequencing

A

Solid phase bridge amplification to clonally amplify templates to generate clusters, followed by single based sequencing using fluorescence

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15
Q

Briefly describe IonTorrent sequencing

A

Measures release of hydrogen ions when a nucleotide is incorporated into DNA by polymerase. The potential difference due to the pH change is converted to a base call by an ion sensor

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16
Q

Name 2 long read sequencing methods

A

Single Molecule Real Time (SMRT) sequencing

Nanopore sequencing

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17
Q

Name an application for SMRT

A

Rapid identification of infectious pathogens

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18
Q

3 steps of nanopore sequencing

A
  1. dsDNA is unzipped to form ssDNA that moves through the pore
  2. Flow of ions produces a current depending on the base in ssDNA passing through the pore
  3. Adaptor molecular keeps bases in place long enough to identify
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19
Q

2 advantages of SRS

A

High accuracy and throughput

Cost effective

20
Q

4 advantages of LRS

A

Allows variant phasing by haplotype generation

Better SV and CNV calling

Can distinguish genes and pseudogenes

Sizing of repeat expansions

21
Q

4 limitations of LRS meaning it is not applicable for diagnostics (yet)

A

Not cost effective

High error rate

Requires high molecular weight DNA

Not standardised

22
Q

4 main steps for NGS bioinformatics

A

QC

Alignment

Variant calling

Annotation

23
Q

3 files for NGS data storage

A

FASTQ (text file with base calls and QC data)

BAM (aligned data with variants/coverage info)

VCF (annotated variants)

24
Q

2 sources of sequencing errors in NGS

A

Polymerase misincorporation

Cross talk from nearby cluster

25
Q

Balance of base calling algorithms should achieve

A

Identify/remove true errors whilst not removing true variants e.g. deletions

26
Q

What are Phred scores?

A

A log converted scale for estimated probability of an incorrect call at a given base

27
Q

What accuracy of base calling does a Phred score of 30 give?

A

99.9%

28
Q

Advantage of pair end reads?

A

More precise alignment especially at repeat regions

29
Q

What coverage is sufficient for diagnostic germline panels?

A

30X with >99% sensitivity for heterozygous variants

30
Q

What scenarios require deep sequencing?

A

Germline disorders showing mosaicism e.g. NF2

Somatic variant detection in samples with low NCC content

31
Q

What coverage is required to accurately identify a SNV at 1% within a sample?

A

5000x

32
Q

Difference between germline and somatic variant interpretation?

A

Germline = is the variant causing the phenotype?

Somatic = is the variant a driver, diagnostic for a specific cancer, associated with good/poor risk or is targetable with a specific drug?

33
Q

Name a pan-cancer database used to aid somatic variant interpretation

A

COSMIC

34
Q

Name 3 sources of targeted NGS panels

A

Custom target enrichment

Clinical exome

Virtual panels for WES/WGS

35
Q

Advantage of virtual panel over custom target enrichment?

A

New genes can be easily added and same data analysed. No re-design of probes required.

36
Q

3 advantages of targeted panels

A

High coverage

Less incidental findings and VUS

Less data storage

37
Q

What % of disease causing variants does WES detect?

A

85%

38
Q

2 advantages of WES

A

Decreased sequencing, analysis and storage costs

Easier variant interpretation (less fatigue)

39
Q

4 advantages of WGS

A

Detects SNV, CNV, indel, UPD, LOH and mosaicism in coding and non-coding gDNA and mtDNA

Increased reliable and uniform coverage (no capture inefficiencies)

No PCR amplification = less GC bias

Less read depth for same coverage

40
Q

What is a gene agnostic testing strategy?

A

Non-targeted panels of many genes (WES?). Used for patients with broad/overlapping phenotypes e.g. dev delay

41
Q

Aim of DDD study and testing strategy

A

Discovery and diagnosis of genetic conditions in patients with severe developmental disorders

Trio WES

42
Q

Diagnostic yield for DDD

A

35%

43
Q

What was the 100kGP and the main aim?

A

Proof of principle study showing the utility of WGS in clinical diagnostics

Aim to create a new genome medicine service for NHS that benefits patients, is ethical based on consent and also enables scientific discovery

44
Q

What was the aim of the PAGE study? 2014-2016

A

Assess the incorporation of WES/WGS in OND and elucidate genetic causes of fetal anomalies. Translate into routine clinical practice.

45
Q

Where was the first rapid WGS service introduced in the NHS? Diagnostic yield and TAT?

A

Wales

41%

TAT = 6-26 days

46
Q

McDermott et al (2022) report summary?

A

Retrospective study of 1 year of rapid WES testing in NHS Birmingham/Exeter labs

Diagnostic yield = 40% (38/95)

Median TAT = 11 days

Altered management in 97%

Flora Ross (12 decks)

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