Revision DNA sequencing and genotyping – 300820 – Genes, Genomics And

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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