20.02.15 NGS - applications Flashcards
What is WGS
- Whole genome sequencing
- Sequencing of entire genome (mtDNA and nuclear DNA)
What is WES
- 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
Pros and cons of WGS and WES
- 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. - 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
Tools for gene discovery
Positional mapping using karyotyping, linkage analysis, homozygosity mapping, CNV analysis, SNP-based association analysis.
Issues that limit gene discovery
- Rare disease loci, often few families/cases to study
- Reduced penetrance
- Locus heterogeneity
- Diminished reproductive fitness.
- Variable phenotype
What are GWAS studies
- 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.
How to prioritize NGS variants for suspected pathogenicity
- Unique in patients or rare in general population
- Located in protein-coding regions
- Affect function of protein encoded by mutated gene
What is targeted NGS and advantages/disadvantages over WGS/WES
- 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
Benefits of virtual panels with exome sequencing
- Reduced likelihood of incidental findings
- Flexible analysis
- Additional genes can be analysed at no extra cost.
Examples of other applications of targeted NGS
- 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.
What is ChIP seq
- Genomic wide profiling of DNA-binding proteins, histone modifications or nucleosomes in vivo.
- Used for studying transcriptional regulation and epigenetic mechanisms
How does ChIP seq work
-Antibodies used to select specific proteins or nucleosomes, which enriches for DNA fragments that are bound to these proteins or nucleosomes
What is RNA-seq
-Sequencing of the transcriptome (complete set of transcripts in a cell) for a specific developmental stage or physiological condition.
What 2 approaches are there for RNA seq
- 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)
Limitations of RNA seq
-Based on Sanger sequencing technology and a significant portion of short tags cannot be uniquely mapped to reference sequence.
How does RNA seq work
- 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.
Advantages of RNA seq
- 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
What is circulating tumour DNA (ctDNA)
- 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.
Use of NGS for HLA typing
-Specific polymorphisms in an individual’s HLA region is essential for organ/stem cell transplantation.
What 3 methods are there for HLA typing
- Sequencing-based typing (SBT)
- Sequence-specific primer (SSP) typing
- Sequence specific oligonucleotide (SSO) typing
What is third-generation sequencing
- 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
What is 100,000 genome project and purpose
-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.
