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Thursday, February 5, 2009

Pacific Biosciences - Steven Turner " Applying Single Molecule Real Time DNA Sequencing"

Realizing the power of polymerase SMRT.
Each nucleotide is labeled uniquely, the flurophores are truncated, leaving behind just the dna. Using a Zero mode waveguide, only the one being read is shown. [cool videos].

At end of signal, it just moves on to the next base...

Every day, they're working on SMRT – showing a demo run.. 3000reaction in parallel. Multi kb genomic fragment. Just one Polymerase. Similar to electrophoresis... keeps going.... and going and going. Real time – several bases per second. Put it to bottom of screen ,just keeps going on and on and on. [it IS transfixing.]

Start with genomic, sheer by any method you want, and now, ligate with HAIRPINS! It's now circular.... so you can keep going around and around. You get both sense and antisense DNA. Can close any size... call it a “SMRTbell prep”, (eg, not a dumbell... heh.. not really that funny.) They also use strand displacement enzyme, so it just displaces what was already there.

First project was a human BAC, last november 107kb chromosome 17. Production readlength: 446bp. Max read: 2,047bp. Aligned to NCBI, and validated by Sanger.

In non repetitive: 99.996% accuracy. Missed 3 SNPs that were false negatives. Repetitive. 99.96% missed 7 bases. Have made significant progress since then..

Sequenced E.coli to 38Fold... 99.3% (last january), max readlength at 2800bases. 99.9999992% [I hope I got that right!]

4 errors + 1 variant on whole genome (Q54!)

Heh.. they had issues from artifact caused by more DNA closer to ORI in E.coli from stopping cultures in midphase. Now have incredibly accuracy that they can measure it.

Accuracy does not vary more than 5% over 1200 bases. Heads for Q60 around 20-fold coverage.

8 molecule coverage. (8 Individual DNA strands have contributed.) Dependant on the fluorophores... they each show brightness profiles. So, some channels are still weak, but they have new ones in development to replace it.

One example: First time you can bridge a single 3200bp region. 3bp/sec. (2.6kb duplication region in the middle.)

Development: average of 946bp read length... and up to 1600 at the high end. You trade throughput with readlength... at one end, fewer SMRT waveguides complete, but long reads, at the other, more complete at the shorter read.

Consensus on a single molecule. You can also do heterogeneity. If you put in mixes, you get out a mix, with a linear relationship to the fraction recovered. (eg, snps will be very clean.)

Flexibility: you can do long OR short reads. Redundancy is high, so you can get 1ppm sensitivity. 12 prototype instruments in operation. Expect delivery in Q3 2010.



Anonymous Anonymous said...

Sequenced E.coli to 38Fold... 99.3% (last january), max readlength at 2800bases. 99.9999992% [I hope I got that right!]

That's on the consensus on those bases with a coverage of x20 or higher.

February 5, 2009 6:27:00 PM PST  

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