Next generation Sequencing Flashcards
Mutation types
Gene mutations
Chromosomal mutations
- number
- structural variants
Mutations involving repetitive DNA sequences
Structural variation (SV)
Structural variation is generally defined as a region of DNA approximately 1 kb and larger in size.
Unbalanced SV ( deletions, insertions and duplication): lead to a net decrease/ increase of DNA —> called copy number variations (CNVs) (e.g. CMT1A involves duplication of PMP22)
Balanced SV (include translocation, and inversion): do not involve a loss of genetic information but may create chimeric or broken genes
Types of treats used to diagnose neurogenetic conditions
Chromosome karyotype and microarrays
(intellectual disability, neurodevelopmental diseases, dysmorphic syndromes)
Biologic assays eg, metabolic assays
DNA- based tests
-single gene test
-NGS gene panel
-whole exome and whole genome sequencing
-test of repeat expansions and other conditions involving repeat DA
-LRS and OGM
NGS sequencing
Step 1: DNA library preparation
(Adapters containing specific sequences designed to interact with the NGS platform, either the surface of the flow-cell (illumina) or beads (ion torrent).
Step 2: template amplification
(Flow cell, lane of the glass slide coated with oligos that are commentary to library adapter, cluster generation occurs on flow cell)
Step 3: sequencing
(4 bases = 4 different dyes, each time a base is incorporated, a colour signal is released, camera images of the coloured bases on the surface of the flow cell every cycle, each image is converted to a nucleotide base call)
Step 4: alignment and data analysis
- process the machine raw data
(Base calling)
- align the short read sequences to the genome
(Alignment)
- call variants in the sequence data
(Variant calling)
- interpret the genetic data
(Variant annotation)
NGS variant analysis
Main filtering steps
1. Inheritance model/ expected segregating variant
- Variant class filtering
- Population frequency filtering
- Assess a subset of candidate/ known causative genes (virtual panels)
- Prioritise candidate variants found
Sequencing and mapping technologies
srWGS (illumina)
Nanopore
PacBio
OGM
