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Thursday, February 5, 2009

Mike Kozal, Yale University, “Ulta Deep Sequencing and Other Genotyping Technologies to Detect Low-Abundance Drug-Resistant Viral Variants”

Lots of political jokes. “Following Eric Lander , I feel like McCain following an Obama speach...”

Talking about three specific viral pathogens: HIV, HCV, HBV.

The percentage of persons surviving longer with Aids is increasing. We're getting better at taking care of these people. Proportion of patients dying with aids is actually aproaching that of a non-infected person. A 25 year old with aids can now be expected to live another 40 years. With all medications, they can halt replication of the desease. The disease burden is still a problem, however.

There are a few patients, however, who have resistant strains. Clearly, this is caused by people who have the disease and getting treatment who are still transmitting. It's about 10% of the population who have resistant strains.

Therefore, in the clinic, HIV genotyping is standard. 1000's are being ordered daily in the US.

In addition to the “sloppy” polymerase, ~10 Billion viruses are produced a day in a single person. (Wow.. no wonder it mutates rapidly! )

Dr. Kozal is now covering some of the tools used for genotyping HIV, as well as how resistance forms.. Highly useful for clinicians, though maybe not so much for those who are already familiar with resistance development. (Re-emergence model.) Clinics still use Sanger based sequencing – so re-emergence is a problem when sequencing can't detect sub-populations.

One major problem in seqeuncing for viruses is linkage: How do you distinguish three separate variants from one variant with three mutations? Could be very important in how the drug treatment is applied.

Note: Oddly enough, at this point, somewhow Dr. Kozal has switched to discussion of amplicons for a sequencing stragegy. I'm a little confused as to what process he used, since this seems to be in the context of 454 sequencing.. I hadn't realized that 454 sequencing required cloning. Or I could be very confused about what he's describing.

Talking about a study done, now, in which some patients failed out quickly – presumably because they harboured a mutation that allowed the virus to dodge the drug being tested. Most of the variations were found at VERY low abundance (<5%) and could not be detected by Sanger seq. Depending on type and level of variant, the patient's time to failure could be predicted.

Switch to Hep B.

Describing Sanger approach to screen, where early studies showed that variants have clinical implications. Main limiting factor is the ability to extract sufficient viral genomes for the assay.

Jumping back to other viruses: apparently 34% of HIV patients who were classified as “wildtype” (by Sanger sequencing) actually have drug resistant strains when the same study is redone with real-time pcr.

Other technologies: PASS technology (parallel-analysis sequencing?) Can get down to 1%, and able to re-analyze the same cluster. Neat – I missed the journal reference though (font was too small - I'll have to move up for the next session..)

Needs: New diagnostic tools – need to move into clinical settings. How sensitive do you need to be for good outcomes? How do you treat linked mutations? Can linkage be used for better outcome prediction. Current floor detection seems to be 0.2% (mainly caused by PCR problems.)

My comment: This is neat, and is certainly an interesting application where Second-Gen sequencing could have a huge impact, yet the talk is mainly about Sanger based sequencing, and how it should be replaced with new technology, with the ultimate question of “how deep do we need to go?” in the study of viral genomics for clinical use.

Edit: Answering questions with "If I could get access to the president's ear..." while Eric Lander is sitting in the audience.  Nice.

(I think I'm going to have to revisit these notes and clean them up at some point... as I look over my notes, I can see they're pretty messy.)



Anonymous Anonymous said...

you're right that 454 sequencing does not require cloning. amplicon sequencing is just that: sequencing PCR-generated fragments/amplicons. the PCR primers are tailed with sequences that allow the resulting amplicons to feed right into 454's emulsion PCR. his talk was primarily about this application i.e. 454 ultra-deep sequencing.

February 7, 2009 7:49:00 AM PST  
Blogger Anthony said...

AH! Thanks. That makes sense. Every once in a while I end up missing a detail while writing. Apparently that was the one that slipped by this time.

I'm also not particularly familiar with 454.. that makes it harder to figure out what's going in the 454 based methodology talks.

February 7, 2009 10:27:00 AM PST  

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