Trigger 9: Genome sequencing Flashcards
outline first generation (sanger) sequencing
1) retrieve genomic DNA
2) fragment DNA
3) clone and amplify
4) sequencing
5) detect
outline second (next) generation sequencing
1) retrieve genomic DNA
2) fragment DNA
3) adaptor ligation
4) amplification
5) detect
third generation sequencing produces
long single molecule reads (over 10,000 bp)
third generation sequencing can sequence
large repeated regions and detect trinucleotide expnsion
what else can third generation detect
epigenetic marks (DNA meth)
disadvantage of third gen
higher per bag error rate than NGS
third generation sequencing is not yet
accurate or cheap enough for whole genome sequencign/ whole exome sequencing
WGS
whole genome sequencing
WES
whole exome sequencing
exome sequencing
sequencing of all coding regions
exome sequencing carries out
10 million ‘reads’ of 75-150 bases per person
what technology does exome sequencing use
second-generation NGS
exome sequencing targets all
coding regions of the genome
how much of the genome is coding
1-2% (30-60MB)
what is the most intensive part of next generation sequencing
data analysis- requires computational skills
why is NGS so powerful for diagnosing rare disease
- most rare diseases have a genetic component
- rare diseases are caused by many different genes
- rare diseases are caused by many different types of mutations
- NGS offers a gene-agnostic approach of testing
what is NGS specifically powerful at
for diagnosing paediatric rate diseases, such as DDs
why is NGS powerful at diagnosing DD
- lots of different genes cause overlapping phenotypes
- reduced reproductive fitness therefore expect ultra-rare variants
- phenotypes arises early, less likely to be influenced by environmental facts
de novo mutations are
easy to find
recessive diseases are enriched in
consanguineous populations
