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
