Bruce Budowle, Federal Bureau of Investigation - “Detection by SOLiD Short-Read Sequencing of Bacilus Anthracis and Tersinia Pestis SNPs for Strain Id
Why do we do this? We've only had one anthrax incident since 2001... but we've been been using bioterrorism for a 2000 years. (several examples given.)
Microbial Forensics. We don't just want knee jerk responses. Essentially the same as any other forensic discipline, again, to reduce risk. This is a very difficult problem. Over 1000 agents known to infect humans: 217 viruses, 538 bacterial species, 307 fungi, 66 parasitic protozoa. Not are all effective, but there are different diagnostic challenges. Laid out on the tree of life.... it's pretty much the whole thing.
Biosynthetic technology. New risks are accruing due to advances in DNA synthesis. Risks are vastly outweighted by benefits of synthesis... bioengineering also plays a role.
Forensic genetic questions:
what is the source?
Is it endemic?
what is the significance?
How confident can you be in results?
Are there alternative explanations?
So, a bit of history on the “Amerantrax” case. VERY complex case, changed the way the government works on this type of case. Different preparations in different envelopes.
Goals and Objectives:
could they reverse engineer the process? To figure out how it was done? No, too complex, didn't happen.
First sequencing – did a 15 locus 4-colour genotyping system. Was not a validated process – but helped identify strain. That helped narrow down the origin of the strain. Some came from texas, but it was more likely to have come from a lab than to come from the woods.
Identifying informative SNPs. Don't need to know the evolution – just the signature. That can be then used for diagnostics. Whole genome sequencing for genotyping was a great use. Back in 2001, most of this WGS wasn't possible. They had a great deal from Tigr – only $125,000 to sequence the genome. From the florida isolate : took 2 weeks, found out interesting details about copy number of plasmids. The major cost was then to validate and understand what was happening.
Florida was compared to Ames to one from UK, which gave 11 SNPs only. Many evolution challenges that came up. The strain they used was “cured” of it's plasmid, so it evolved to have other SNPs... a very poor reference genome.
The key to identification: one of the microbiologists discovered that some cultures had different morphology. That was then used as another signature for identifying the source.
Limited Strategy: it didn't give the whole answer – only allows them to rule out some colonies. It would be more useful to sequence full genomes... so entered into deal with ABI SOLiD for genome sequencing.
Some features were very appealing. One of them is the Emulsion PCR. Helped to improve quality and reliability of the assay. And beads, were useful too.
Multiplex value was very useful. Could test 8 samples simultaneously using barcoding, including the reference Ames strain. Coverage was 16x-80x, depending on DNA concentration. Multiple starting points gives more confidence, and to find better SNPs.
Compare to reference: found 12 SNPs in resequenced reference. When you look at SNP data, you see that there was a lot of confidence if it's in both direction... however, it only turns up on the one strand. That becomes a major way to remove false positive result. That was really only possible by using higher coverage.
Not going to talk about Pestis.. (almost out of time.) Similar points, 130-180X coverage. Found multidrug transporter in the strain which has been a lab strain for 50 years. Plasmids were also higher coverage. SNPs were less in the north american, etc.
An interesting point. If you go to the ref in genbank, there are known errors in the sequence. Several have been corrected, and the higher coverage was helpful in figuring out the real sequence past the errors.
$1000 /strain using multiplex, using equipment that is not yet available. This type of data really changes the game, and can now screen samples VERY quickly (a week).
Every project is a large scale sequencing project
depth is good
multiplexing is good
keep moving to higher accuracy sequencing.
Labels: AGBT 2009