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Next Gen Sequencing - 4 steps
Do not rely on chain termination
1) template strand is immobilized
2)amplified into large number of identical fragments
3)reagents added
4)sequenced by synthesis
-hundreds/thousands strands are sequenced in parallel
Main advantages of NGS over Sanger (5)
-Cost - human genome sequenced for 5K
-Sample Size - less template required
-Speed - massively paralleled
-Accuracy - sequenced multiple times
-not limited to organisms with published genomes
NGS includes
a number of diff modern sequencing techs (6 in slide)
Roche 454 Pyrosequencing
**need clarification
-fragments are attached to bead
-added to well
- the light signal released is correlated with the number of nucleotides joined to the growing strand (ex: GG is brighter than G)
Life tech ion torrent sequencing
-DNA is fragmented
-added to bead
-amplifyed by PCR
-placed in single well of slide
-slide is flooded with dNTPs, releases H ions, decreases pH
-pH is detected
Illumina Sequencing
**Need clarification
slide is flooded with flouroscently labelled nucleotides
-image is taken of each "cycle"
-fluorescent signal indicates which base has been added
5 applications of NGS
1. Agricultural Genomics
-improve drought tolerance of corn hybrids
2. Cancer Genomics
-sequence normal tissue, to identify mutations in tumour tissue
3. Forensic Genomics
-disaster victim identification, complete info from highly damaged samples
4. Complex disease genomics
-not all diseases are associated with one gene - detect genetic variants to multiple samples
5. Microbial Genomics
-sequencing bacteria
International Cancer Genome Consortium (ICGC)
What: large scale caner genome study for 50 diff cancer types
Rationale:
-Scope: huge, no country can do it all
-Reduce duplication
-Standardization and Uniform Quality
-Accelerate dissemination
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