Applications of NGS in Molecular Pathology of Cancer Flashcards
What does cancer genomics refer to?
The analysis of genes and how alterations in genes can lead to changes in cells which cause cancer
What contributes to the development of human tumours?
Genetic and epigenetic mechanisms
In cancer gene alterations can occur at the chromosome level, at the genetic level or at the epigenetic level.
What are some examples?
Chromosomal variants (SVs) – translocations, inversions, duplications, deletions, amplifications (CNVs)
Gene variants – point mutations (SNVs), copy number variations (CNVs), small indels (insertions and deletions)
Epigenetic variants – DNA methylation, histone modifications
What is the clinical relevance of cancer genomics?
Genomic analysis can provide a better understanding of the types of mutations in a tumour
Molecular classification of tumours
Identification of individual targetable genomic alterations
Understand tumour mutation burden (TMB)
Assess tumour heterogeneity
Characterise mechanism of resistance and tumour evolution
Identify complex mutation signatures and cancer specific neo- antigens
Monitoring treatment response
Lead to improved ways to diagnosis, treatment and ultimately better patient care
NGS was developed with the concept of what?
Massively Parallel Sequencing -
All MPS platforms require a library with custom linkers (adapters) attached to the ends of the fragments
Each library fragment is amplified on a solid surface – bead or a flat surface with covalently attached adapters that hybridize to the library adapter
Direct step-by-step detection of the nucleotide base incorporated
Hundreds of thousands to hundreds of millions of reactions sequenced per run
- What are examples of massively Parallel Sequencing?
2. What are the two most dominant?
- Roche/ 454 Pyrosequencing
Ion Torrent
Illumina/Solexa Sequencing
ABI/SOLID Sequencing
- Ion Torrent
Illumina/Solexa Sequencing
What steps are involved in Ion torrent sequencing?
Ion GeneStudio S5 System/ Ion Torrent Genexus System
Library Prep
Emulsion PCR
Ion semiconductor sequencing
What are the pros of Ion torrent sequencing?
What are the cons?
Fast
Error rate of 1%
Suitable for clinical diagnostics
Difficulty of interpreting homopolymer sequences (>6 bp)
What is the NGS workflow for Illumina sequencing?
Sample acquisition and DNA/RNA
extraction
Library preparation
Sequencing
Alignment to the reference genome
Variant discovery
What does quality control sample acquisition for DNA/RNA extraction involve?
Samples Sources: Cells Tissues Blood Biological fluids Fresh Frozen Fixed FFPE Laser capture
Methods for QC: Agarose gel Nanodrop (260/280nm, 260/230nm) Qubit Bioanalyser (RIN) Tapestation (DIN)
Genomic DNA samples should be free of PCR inhibitors (high concentration of EDTA, salt)
DNA samples must not be contaminated with other sources of DNA or RNA
Any contaminant DNA will be amplified and affects the data
Accurate quantitation is critical for any downstream applications
What tools are used to conduct quality control?
Check Quality (Bioanalyzer Tapestation): - DNA and RNA integrity can be measured - DIN and RIN
DIN- DNA integrity number
1= highly degraded, 10= highly intact DNA
DIN >8.0
DNA >50 kb
RIN >7.0
RNA is DNA free
DNase treatment
Check Purity
(Nanodrop):
260/280 ~ 1.8 for DNA,
lower ratio = protein
contamination
260/280 ~2.0 for RNA,
lower ratio = protein
contamination
260/230 ~2.0 - 2.2 lower or higher ratio = organic contaminants
Purify sample
Re-confirm purity
Check Quantity (Qubit):
Meet input spec of your downstream application
Two close readings for each sample
Sufficient leftover for validation
Consider drying sample Consider alternate suitable for sample
Why conduct sample QC?
Quality of sample determines quality of data attained using sequencing
