Next Generation Sequencing Technologies文档.pptVIP

* * * * * * * * The goal is to amplify millions of “polonies” on a single slide. But then we want to perform enzymatic manipulations on this DNA To facilitate this, one of the primers used in the PCR has a 5’ acrylic group that allows copolymerization into the gel. * Now we’ve amplified polonies, How are we going to sequence them. Here’s how we do this. * The first thing I want to ask is can we amplify single molecules at high densities in acrylamide? Alexander Chetverin had previously demonstrated growing RNA colonies in agarose using Q beta replicase system so we were optimistic that DNA colonies could be grown. Add various amount of templatre (1 kind 236 base pairs), amplify, stain with DNA DYE SYBR GREEN - see fluorescent spheres? Number of spheres linear with amount of template added Pick sphere and run on gel – right size. Conclude that these spheres were DNA colonies or polonies. Now the size of the polonies shown here is much larger than we would like. We could fit 2100 of these polonies on a single slide and we want millions. * But, by increasing the length of the DNA template and increasing the acrylamide concentration, we can get polonies as small as 5 microns. This would enable 15,000,000 distinguishable polonies assuming Poisson statistics. * We established that we could amplify polonies But can we sequence them? Rather than sequencing them directly, I decided to try to work out the protocol using oligonucleotide templates attached to acrylamide to work out issues with attachment chemistry, as well as the nucleotide and polymerase chemistries. First question, can we get a specific single base extension with our * * * * * * * * * Map further away, then closer * * * * * * * * * * * * * * * * * * How did they do? 150 bp circular template ~93% raw accuracy 15x coverage 99.3% accuracy Still early days Where are they going Phi29 so long read lengths possible Ease of sample prep Camera costs Summary Sequencing will become very inexpensive