Lecture 3 - Microarrays Flashcards
What do sequencing and microarrays allow you to do?
Read sequence of DNA/RNA or epigenetic modifications
Sequencing vs Microarrays
Sequencing - measure whole sequence of DNA
Microarrays - measure large number of genetic variants simultaneously
3 advantages of sequencing
greater coverage
can identify novel gene variants
unbiased
disadvantages of sequencing
expensive and very complex analysis
3 advantages of microarrays
cheap
well validated analysis methods
relatively low amount of input DNA
3 disadvantages of microarrays
limited to a certain number of known loci
depends on prior sequence knowledge
biased
Application of Whole Genome Sequencing in Cancer
discovery of mutations, mapping of structural
rearrangements
Transcriptome analysis (RNA-seq) application cancer
sequencing totalRNA, mRNA or small RNAs
Methylome analysis application cancer
classification of tumours, biomarker discovery
ChIP-seq application cancer
to profile chromatin marks DNA-protein interactions
across the genome
Whole genome sequencing steps: 3
Break DNA into short fragments
Repair ends w/ adaptors
Sequence
Whole exome sequencing 5
Break DNA into short fragments Repair ends w/adaptors Capture fragments containing exons Wash uncaptured DNA Sequence
RNA sequencing 8
Remove contaminant DNA remove rRNA Fragment RNA Reverse transcribe into cDNA Ligate sequence adaptors PCR Select a range of sizes Sequence cDNA ends
Methylation Analysis Steps: 5
Bisulfite Conversion of genomic DNA
Random primed DNA synthesis of ssDNA fragments
3’ tagging
PCR
Sanger Sequencing 4
DNA fragmentation
In vivo cloning and amplification
Cycle sequencing : incorporation of fluorescent tagged chain terminating
dideoxynucleotides during replication
Order of bases determined by size of fragment
Illumina Sequencing
DNA is attached to a flow cell
Denaturation
Bridge amplification
Sequence is built up over multiple cycles involving fluorescent labelling to see which base is in each cluster
Polysequencing and Ion torrent
“Sequencing by synthesis” principle
When nucleotides are incorporated, detects
release of pyrophosphate
Intensity of light determines number of same
bases in a row
Nanopore 2
Nanopore (tiny hole – 1 nanometer) immersed in
conducting fluid with charge across it
Nucleotides passing through the hole creates
change in current from which sequence can be
measured
