Next gen sequencing:
Next-Generation DNA
Sequencing Methods
Elaine R. Mardis 2008
- blunt end ligate adaptor sequences to DNA fragments
- these adaptors can be used to PCR amplify the DNA, eliminating the requirement for a time consuming plasmid amplification step
- long read times: 8hrs to 10 days (shitloads of microreads of 25-30bp): each run could yield 10s of millions of sequence reads eg Illumina system or Applied Biosystems SOLID
- due to long read times without operator intervention and reduced preparation reqs, labour intensity = very low

Pacific biosystems:
- 'human genome in minutes for under $100'
- multiplex of ZMW fluoroscopy microcells: phosphonucleotides (4 different fluorophores attached to 3' phosphate group) are added to ZMW chamber with DNA polymerase.
- when a NT is added by DNA Pol, flurophore is excited and the emission spectrum measured by ZMW to determine which NT was added

Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

Chromatin Immunoprecipitation: Look at epigenetic properties of genes (eg whether a particular gene is bound to histones and thus unlikely to be expressed): Crosslink DNA and DNA binding proteins using Formaldehyde; break the DNA apart by sonication; Add an antibody specific to the DNA binding protein (eg histone); run it all through an antibody affinity column; and tada: you have all the DNA that is bound to a particular DNA BP. Using next gen sequencing, this DNA can then be sequenced to find out which genes are affected by epigenetical modifications.

SAGE: (Serial analysis of Gene expression):
- SAGE can monitor levels of gene transcription by looking at mRNA levels
- each mRNA transcript can be adequately codified by a 'tag' that is composed of 26 nucleotides (SUPER SAGE using type III-endonuclease EcoP15I of phage P1) long enough for the sequence to be pretty much unique
- so you tear a bit off each mRNA (or more correctly cDNA) molecule and then, (and here's the beauty of the technique), you ligate all the tags together to form one long DNA strand and then sequence it! Heaps easier than PCRing each individual cDNA!

Ancient DNA sequencing:
- because NGS only requires single DNA molecules, degraded DNA from bones can be sequenced
- neanderthal genome is being sequenced through this method

Metagenomics:
- high throughput, low cost is making metagenomic studies possible.
- Can look at RNA present in a particular site (eg. acid mine) and look for genes involved in metabolisation of toxic/waste products