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Friday, February 26, 2010

AGBT 2010 -Pacfic Biosciences Workshop

"The debut of the 3rd Generation"

* Came from the basement of a building in cornell. [what is it with basements on campus?]
* technology detects 500 photons per base
* Raised $266M in company history

* show slide with first results that launched company - detecting 3 labeled C's, barely

"yes, it is big, yes, it is heavy, and yes, it does work"
* smallest: $50,000 desktop version
* Largest: full human genome in 15 minutes.

Already have manufacturing for reagents - and building a facility to construct machines.

Steve Turner: Founder, CSO, Board Member

1. brief overview of technology
2. Update on Collaborations
3. Instrument debut
4. Applications
5. Scalability

* Video of polymerase - same one from web.

* influenza
* cancer transcript
* long read progress
* strobe seqeuncing for strutral variation
* Palustris systems biology [Go palustris!!!]
* circular sonsensus sequencing
* survey of coverage bias
* direct detection of methylation and DNA modification

* serotyping doesn't give picture - immunologically distict viruses.
* Fast Time to result: 9 hours from sample Extraction to sequencing analysis completion.
* did not look at consensus call - used single molecule reads.
* match single molecules with sequenced refernce genomes of similar influenza.
* Turned out that the strain was misidentified - phylogeny was incorrect.
* side benefit: in every case, each segment was covered in single reads. Potential for quasi-species studies of viruses.

Sequenced MCF-7
* known alt. splice forms implicated in tumorigenesis.
* Can map entire transcripts (2400bases) in single read.
[neat stuff]

10,351 base read scrolling... goes on and on.
* they see up to 20kb reads.

Strobe sequencing
* answer to Mate Pairs?
* Polymerase is damaged by laser, so reads will continue until damaged
* Turn off the light, and the polymerase is unharmed... will continue till you turn the lights back on.
* Who needs mate pairs when you can just sequence 10kb at a time?
* show repeat lengths - at 20kb, you can sequence most of your repeat regions. - Strobe it as well...
* Very useful for assembly.

Insertion AC223433 fosmid
* can use time as a way to look at insert size.

* 58 contigs from palustris
* Hybrid assembly - now have a single contig. (Used Strobe, straight and other tech..)

Read Length.
* Expect that you can epxand readss to 50-70kb.
* demonstrate by haprpin ligation to lambda genome (linear)

circular consensus sequencing
* make something circular, then go 'round and 'round till you get consensus.
* Q40 on single molecules by going over it many times

* results in Low bias for GC content
* tested on many organisms

modified nuclear bases
* look at kinetics of base incorporation
* modified nuclear base Methylated Adenosine causes kinetic differences
** 6-10x kinetic changes.
* Methylated Cytosine - still get a signal
* Hydroxymethycytosine: can also see that - also different from other traces
* duration and spacing are different for the three bases.
* Single base resolution, less than 1% FP, methylation detection on single moleucles
* also looked at other modifications - can always tell that it's different.
* Polymerase stalls at T-dimers.

[Summarized it all]

[Insert CEO talk here - wonderful company, wonderful people, "state of the art", hard work.]

Unveil worlds first 3rd gneration sequencer
* Movie time!
* 8 Cells per package - $100 per cell.
* SMRT Cell - 96 / tray.
* reagent plate (96 well)
* each cell works indepenently - in any protocol
* Uses CSV files
* API to LIMS with designs.
* System looks pretty child-proof (though probably not idiot-proof)

Monitoring ar run:
1. monitor at instrument or remotely
2. View real time base incorporations
3. remaining runtime
4. status of each cell from cell prep to run.

Signal to noise ratio is dramatically improved from last year


* web based interface
* accessible from any computer
* automated secondary analysis

* full complement of reports automatically generated
* quality files
* ....

Browswer integrated into viewer.

* FastQ
* etc...

All in one day.
* sample prep to analysis.

* methylation sequencing will be released in an update
* direct rRNA sequencing.

Working towards SMRT Translation
* replace Trancription (Polymerase) with translation (ribosome & labeled tRNA....)

[ok, didn't see that coming]

Scaling of performance over instrument life
* current yeild 30% improved to 90%
* Multiplex: 80k improves to 160,000
* speed 1-3bps improving to 15bps

Throughput should pass 2nd generation with this instrument. Expect new instrument in 3 years to blow all of this away.

Interpretation of Genomics will require epigenetics, etc etc etc. and much data processing. [Oddly, That's what I tried to convince Complete Genomics people of this morning, without success.]

* Dark Bases? They are not dark bases - they are missed bases. They now have better bases, that bind better than the natural bases. Missed bases are a problem - the nucleotide docks, and if happens too fast, you don't get enough phototons...

* Something about algorithms for de novo assembly - check out the posters, and we'll have more information for you.

* What is your error rate? [Very agressive question] Single pass error rate is greater than ensemble sequencing. You don't get systematic error in Pac Bio - Approach towards consensus is linear. You know when you see systematic errors - you can catch and repair. Expect Q90 with this technology.

* Exponential decay on read lengths.



Anonymous Anonymous said...

What was the general perception of Life technologies 3rd Generation sequencing presentation?

February 27, 2010 12:32:00 PM PST  
Blogger Anthony Fejes said...

Actually, I don't know - and I'd hate to try to give the general perception on anything. I talked with a lot of people at AGBT, but I really don't think I've talked to enough to get a reasonable sample.

In fact, that was one of the few workshops I missed - I confess, I was enjoying the sunshine outside (All 20 minutes that I managed to get.) during that workshop.

However, all of us canadians were hanging out in the Life Technologies suite for one of the hockey games, and I managed to spend some time watching their video earlier on the new technology.

Really, though, the rep was more interested in chatting with someone who's much better looking than I was - so I failed to understand how their technology is different from an Illumina sequencing run. (Single Molecules, instead of clusters and a fluorophore of some sort tagged to the enzyme, perhaps?)

Anyhow, If anyone has anything to add, I'd be happy to post it.

February 27, 2010 1:37:00 PM PST  

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