GGHH 300820 2021
Workshop: DNA and RNA sequencing and Genotyping
Why do we need to sequence the human genome?
Why do we need to sequence genomes from different populations?
DNA Sequencing
- Targeted DNA sequencing (Sanger Sequencing): sequencing small
targeted regions of the genome to tests for the presence/absence of
mutations (as well as many applications in molecular genetics research
laboratories) - Whole-genome sequencing (Next generation or massively parallel DNA
sequencing): sequencing of whole genomes, or all exons in a genome
(exomicDNA sequencing), essential for cataloguing rare and common
genetic variants across the human genome
Summarise the methodology of Sanger DNA sequencing
The Sanger DNA sequencing method uses di-deoxynucleotides
- The absence of the 3’ hydroxyl group prevents
incorporation of a subsequent nucleotide - The incorporation of the 2’, 3’ di-deoxynucleotide
effectively terminates elongation of the DNA
chain by DNA polymerase
X
Third Edition © 2019 Elsevier Inc. Molecular Biology
4 di -deoxynucleotide triphosphates (ddNTPseach carry a different fluorescent molecule; DNA )
polymerase extends the primer and incorporates the ddNTPs
DNA fragments are separated by highcapillary electrophoresis, laser light excites the -resolution
labelled dNTPs
Light is read by a detector and a sequencing trace (electropherogram) appears on the
computer
Incorporation and sequence termination by complementary ddNTPsresults in a
pool of labelled fragments
TT
AG
GA
CC
Sanger DNA Sequencing
Nature Reviews Neuroscience 13, 4531038/nrn3271 -464 (July 2012) doi:
Fragmentation of DNA
Add adaptors
Add probes that are specific for exons across
the human genome
Capture exongenomic DNA fragments-containing
An adapter is a short double-stranded DNA
oligonucleotide that is attached to the ends of
the genomic DNA using the enzyme DNA
ligase.
Genomic DNA is fragmented using
ultrasound
Sequencing by Synthesis (Illumina)
Define coverage
How does whole genome/exome DNA sequencing differ from whole -genome genotyping?
AGCTAGAGTGAGCCTTAGTAAGACATCGATCTCGACTGGAATCATTCTGT
Oligonucleotide Sequences flanking the tagging SNP are
immobilized onto the chip
1
Fragmented and denatured genomic DNA is hybridized
to the chip
TCGATCTCGACTGGAATCATTCTGT AGCTAGAGTGAGCCTTAGTAAGACATCGATCTCGACTGGAATCATTCTGT
CCCAACAC
…
AGCTAGAGTGAGCCTTAGTAAGACA
TGGTTGTG
…
2
A single complementary base labelled with a fluorescent probe
is incorporated
AGCTAGAGTGAGCCTTAGTAAGACA
G
TCGATCTCGACTGGAATCATTCTGT
A
TCGATCTCGACTGGAATCATTCTGT
CCCAACAC
…
AGCTAGAGTGAGCCTTAGTAAGACA
TGGTTGTG
…
3
GWAS SNP Genotyping: Summary
Differences in the emitted wavelength of fluorescent light is used to determine the
genotype
How has the 1000 genomes project contributed to GWAS genotyping projects?
Describe a scenario that would require the use of
whole exome and Sanger DNA sequencing
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