Topic 5b: technologies in genetics I Flashcards
what was the first method used to sequence the human genome?
sanger sequencing
how does sanger sequencing work?
- Take DNA, and using a primer of a known sequence, use DNA polymerase along with fluorescent ddNTPs (which will make it terminal) and normal dNTPs,
- and with that we will get sequences of random size of sequences that will end with a fluorescent nucleotide,
- which is separated with capillary electrophoresis, and looking at the colour, we can determine which base it is.
what are general sequencing principles to make a library prep?
- take genomic DNA
- fragmentation
- end-repair
- dA tailing
- adapter ligation
what is a disadvantage of sanger sequencing?
very expensive and time consuming
how does the illumina sequencing technology work?
- take known DNA and fuse it to unknown DNA
- on a microscope slide, induce cluster growth (amplify a sequence in clusters) – each sequence will find their “partner” on the slide, forming a bridge
- cycle sequencing the clusters via fluorescence, using a primer (of the known DNA from before) with only fluorescent ddNTPs and add 1 at a time and image it after each addition, then we can figure out the sequence
- then with the image we can figure out what the bases are of the cluster
how does de novo sequencing work to construct the genome if we don’t have a known sequence vs when we have a known sequence?
unknown: take sequence reads, overlap them and then you get a linear sequence of the gene/sample
known: take small sequence reads and compare them to a known sequence, and if we see the same difference in many samples/sequences, we can say with confidence that there is a change and it is not an error
what variations can we detect with sequencing?
SNPs, small deletions, CNVs, and chromosomal abnormalities
how does whole exome sequencing differ and what is its purpose?
only sequence the exons (the coding part of the genome) and is used for clinical or molecular diagnostics – in order to make it cost effective
what is the exome-seq workflow?
- take genomic DNA
- fragmentation
- make a biotinylated RNA library
- hybridization
- use bead capture to “fish-out” the coding regions
- digest RNA
- sequencing
- analysis
what are some disadvantages of exome sequencing?
- Analyzing only rare variants in coding regions is
not always conclusive. - Variants in non-coding regions and CNV have also
important value and should not be ignored
what are some disadvantages of whole genome sequencing?
- Identifying all SNPs ( order of 10 million)
- Resolving Haplotypes and phase is not always straight forward
with short reads - Identifying structural variation (4.8-9.5%/genome)
- Overcome segmental duplications
- Complex regions (HLA, KIR etc)
-Resolve trinucleotide repeats, microsatellites - Sequencing process takes >12h
