20.02.15 NGS - applications

Question 1

What is WGS

Answer 1

• Whole genome sequencing

- Sequencing of entire genome (mtDNA and nuclear DNA)

Question 2

What is WES

Answer 2

• Whole exome sequencing
• Covers coding sequences of annotated protein-coding genes (23,000)
• 1-2% of total haploid genomic sequence (30Mb)
• Contains 85% of DNA mutations that have an effect on human disease

Question 3

Pros and cons of WGS and WES

Answer 3

1. Pros of WGS
   - WGS can detect SNVs, indels, SV and CNVs in coding and non-coding regions
   - WGS has more reliable/uniform coverage at an average lower read depth.
   - WES has capture and amplification bias
   - Exome is changing as more target exons are identified, means older capture kits won’t target them.
2. Pros of WES
   - Cost effective to sequence protein coding regions. Lower storage and analysis costs.
   - Reduced costs means more samples can be tested. Larger population based comparisons possible

Question 4

Tools for gene discovery

Answer 4

Positional mapping using karyotyping, linkage analysis, homozygosity mapping, CNV analysis, SNP-based association analysis.

Question 5

Issues that limit gene discovery

Answer 5

• Rare disease loci, often few families/cases to study
• Reduced penetrance
• Locus heterogeneity
• Diminished reproductive fitness.
• Variable phenotype

Question 6

What are GWAS studies

Answer 6

• Genome-wide association studies
• Used to discover loci involved in complex disease.
• However, loci collectively account for a small fraction of the observed heritability of the trait of interest.

Question 7

How to prioritize NGS variants for suspected pathogenicity

Answer 7

• Unique in patients or rare in general population
• Located in protein-coding regions
• Affect function of protein encoded by mutated gene

Question 8

What is targeted NGS and advantages/disadvantages over WGS/WES

Answer 8

• Disease-specific targeted tests
• Advantages over WGS/WES: cheaper, coverage is better as can gapfill with Sanger, easier interpretation, less chance of incidental findings.
• Disadvantages: inflexible design

Question 9

Benefits of virtual panels with exome sequencing

Answer 9

• Reduced likelihood of incidental findings
• Flexible analysis
• Additional genes can be analysed at no extra cost.

Question 10

Examples of other applications of targeted NGS

Answer 10

• Tumour profiling: deep sequencing so higher sequencing, parallel testing of multiple samples, shorter TATs possible
• Monitoring minimal residual disease (MRD): able to detect emergence of clonal dominance (allelic ratio determined for sequence variants)
• Prenatal testing/screening: NIPT/NIPD, PGD
• Clinical microbiology: bacterial genomes can be sequenced to provide information on resistance, virulence and typing during outbreak investigations. Need short TAT.

Question 11

What is ChIP seq

Answer 11

• Genomic wide profiling of DNA-binding proteins, histone modifications or nucleosomes in vivo.
• Used for studying transcriptional regulation and epigenetic mechanisms

Question 12

How does ChIP seq work

Answer 12

-Antibodies used to select specific proteins or nucleosomes, which enriches for DNA fragments that are bound to these proteins or nucleosomes

Question 13

What is RNA-seq

Answer 13

-Sequencing of the transcriptome (complete set of transcripts in a cell) for a specific developmental stage or physiological condition.

Question 14

What 2 approaches are there for RNA seq

Answer 14

• Hybridisation-based: incubating fluorescently labelled cDNA with custom made microarrays
• Sequence-based: Sanger of cDNA, massively parallel signature sequencing (MPSS), serial analysis of gene expression (SAGE)

Question 15

Limitations of RNA seq

Answer 15

-Based on Sanger sequencing technology and a significant portion of short tags cannot be uniquely mapped to reference sequence.

Question 16

How does RNA seq work

Answer 16

• Population of RNA is converted to a library of cDNA fragments with adaptors. Each molecule is sequenced (with or without amplification) with single or paired-end sequencing.
• Reads are then aligned to reference genome/transcripts or assembled de novo.
• Forms a transcription map that consists of both the transcriptional structure and/or level of expression for each gene.

Question 17

Advantages of RNA seq

Answer 17

• Not limited to detecting transcripts that correspond to existing genomic sequence.
• Reveals precise location of transcription boundaries (single-base resolution)
• Reveals sequence variations (SNPs in transcribed regions)
• Accurate expression level quantification
• Little sample required

Question 18

What is circulating tumour DNA (ctDNA)

Answer 18

• NGS applied to sequence ctDNA
• ctDNA can acts as a tumour biomarker to identify cancer recurrence, MRD, prognosis, treatment selection
• Higher sensitivity than a tissue biopsy.

Question 19

Use of NGS for HLA typing

Answer 19

-Specific polymorphisms in an individual’s HLA region is essential for organ/stem cell transplantation.

Question 20

What 3 methods are there for HLA typing

Answer 20

1. Sequencing-based typing (SBT)
2. Sequence-specific primer (SSP) typing
3. Sequence specific oligonucleotide (SSO) typing

Question 21

What is third-generation sequencing

Answer 21

• Real time sequencing-by-synthesis technology.
• Avoids PCR-based artefacts, longer read length (easier genome assembly and detection of SVs)
• Can detect RNA/DNA base modifications

Question 22

What is 100,000 genome project and purpose

Answer 22

-Launched 2012.
Objective= to sequence 100k genomes from NHS patients and families with rare diseases and cancer.
-Delivered by Genomics England a Department of Health owned company.