Fourth Annual DNA Grantees' Workshop
Monday, June 23, 2003
AFTERNOON SESSION
Research Update Briefings: Prototypes and Products (continued)
Christine S. Tomsey, Moderator
Biography
MS. TOMSEY: Could I have your attention, please. In the interest of time I ask you to resume your seats so that we can get underway with this afternoon's presentations. I hate to be the one to awaken you from your meditations, but we do have a very distinguished panel of researchers and practitioners for this afternoon.
I am Christine Tomsey, the DNA laboratory technical leader with the Pennsylvania State Police DNA Laboratory, and I would like to first take this opportunity to thank NIJ for these wonderful accommodations and an excellent lunch. It's good to see our tax dollars are at work in this manner.
NIST Y-Chromosome Work and Standard Reference Material 2395
John M. Butler
Biography
MS. TOMSEY: The first individual that I'd like to introduce this afternoon is Dr. John Butler. We've only had one morning for this grantees' workshop, and already I think Dr. Butler's name has been mentioned 16 times, all in a very good vein, I might add. John is known to most of us as an idea man. At any of the Scientific Working Group on DNA Analysis Methods (SWGDAM) meetings and workshops, we always look to John for his opinion. He's done wonderful work with multiplexing.
He started out with his dissertation at the FBI Laboratory, Forensic Science Research and Training Center, with two other rather important idea men: Bruce McCord and Bruce Budowle. John has worked with both of these individuals on capillary electrophoresis for DNA typing involving STRs (short tandem repeats) and mitochondrial DNA.
After the completion of his dissertation work, he joined the National Institute of Standards and Technology (NIST), where he did his postdoctoral work with Dennis Reeder, who I think we all know is Dr. STRBase, and developed the STRBase Web site. In addition to that, he went to GeneTrace Systems in California, where he was a staff scientist and project leader on time-of-flight mass spectrometry for STRs and SNPs (single nucleotide polymorphisms).
In fall 1999, Dr. Butler rejoined NIST, where he's currently a research scientist and principal investigator for the National Institute of Justice on funded projects for future technologies in forensic DNA typing.
Now, I know all of us are very familiar with all of the publications that Dr. Butler has given us—more than 25 books, chapters, and publications—and I think all of us are well aware of his recent textbook, Forensic DNA Typing. All of us have used it in one way or another to help educate our prosecutors and especially to train our new forensic scientists.
DR. BUTLER: Thank you for the kind introduction. Today I'm going to talk about the Y-chromosome work we've been doing in finishing up with the standard reference material (SRM) that we have just completed, SRM 2395. Just to point out the currency of this work, the Y chromosome was just on the front cover of this past week's issue of Nature, in which the full sequence of the Y chromosome is presented. The interesting thing about it is that it's much more complex than we had previously thought. I encourage you to go and read it. Butler: Slide 1
Today I'm going to talk a little bit about what core loci are being used right now for Y-short tandem repeats (Y–STRs); what commercial kits are available or will be becoming available for Y–STRS; some assays that we have worked on, including the "manly-plex" that Lisa Forman mentioned; and the NIST SRM. Butler: Slide 2
Why would we use the Y chromosome? I'm sure we'll hear a number of other talks later about this, but I believe the Y chromosome is much easier to use in forensic casework. For example, in sexual assault evidence you have a male-specific amplification that is made possible by looking at the Y chromosome. Butler: Slide 3
Of course, there are a number of other things: paternity testing, missing persons, migration studies, and genealogical research, which is starting to take off, where you can trace family lineages using the Y chromosome.
Within the forensic community, there's a core set of loci that are being used. Here, on the left, we have the minimal haplotype. There are nine different loci—well, it depends on how you number them, but this locus here (385) has two copies on the Y chromosome, so we call it A and B. With one primer set, you can get these two amplicons, and these are the ranges of known alleles of them and the repeat motifs. So most of them are tetranucleotide repeats, with the exception of 392, which is a trinucleotide repeat. Butler: Slide 4
In Europe, they've used the extended haplotype for a number of years, and this includes a dinucleotide repeat (YCAII), which has two copies on the Y chromosome. More recently, SWGDAM selected the U.S. haplotype, which is the minimal haplotype from the original European group, and a new pentanucleotide 438 and a tetranucleotide 439. This was defined by SWGDAM's subcommittee on the Y chromosome back in January of this year [2003].
We're not that interested in YCAII because you have a high degree of stutter with dinucleotide repeats, as you know, and that's why we use tetras in the forensic community. With YCAII, you have about 50 percent stutter. It's a very nice polymorphic marker, but it's hard to type if you're dealing with mixtures in forensic situations. Butler: Slide 5
The pentanucleotide 438 has less than 5 percent stutter, and 439 has less than 10 percent stutter, so they can handle mixtures much better.
We've done an extensive population study that's going to be published soon in Forensic Science International that looks at how the extended haplotype works versus the U.S. haplotype. As you can see here, with some African-Americans, you get almost the same power of discrimination between the extended haplotype—essentially just the YCAII marker—versus the U.S. haplotype, which is 438 and 439 with the minimal haplotype. Butler: Slide 6
But you can also see that you don't get 100-percent discrimination between these individuals, which is just like mitochondrial DNA, because you have possibilities of closely related family lineages.
So, the bottom line is the U.S. haplotype, the new markers that were just selected, works just about as well as the extended haplotype in all major U.S. populations. In terms of being able to type these, currently the only two kits that are commercially available are the Y–plex 6 and Y–plex 5 from ReliaGene, and those two amplifications together give you the entire 11 core loci for the Y–STRs. Butler: Slide 7
Promega has the prototype that should be out very soon that includes in one amplification all core loci plus this 437 locus and a single amplification. Here are the allelic ladders from their prototype. Butler: Slide 8
We've been developing a number of multiplex assays on the Y chromosome at NIST. This illustrates four of the ones that we've published. Here is the 20plex that was published last September, and these new ones will be in the current publication that will be coming out soon. We published on this 10plex a while back, and it has been adopted by Orchid. Cellmark used it quite a bit in their testing. This just illustrates a number of the primers that are present in the kits because you have some multicopy regions of the Y chromosome that you're amplifying with a single set of primers. Butler: Slide 9
This is the recently published paper on the 20plex amplification. Butler: Slide 10
This is the manly-plex that Lisa Forman was referring to earlier. We typed this relative to the kits to verify that we get the same results. The 20plex is on top, and ReliaGene's 6plex is on the bottom. You can see that the 385 gets the same type 16, 17; and 389II, 390, 391, 393, and TYS19 are also present in both the NIST 20plex and Y–plex 6. So those are all the loci. We get fully concordant results on over 200 samples that we compared directly with the Y-plex 6 kit versus our multiplex. Butler: Slide 11
In terms of how to build these multiplexes, we published a paper a few months ago that describes the entire process. The first part involves very careful primer design. We've written some software that allows us to do that, and we have stringent primer quality control. Also, we've published in both these areas to illustrate how you design and build these multiplexes. Butler: Slide 12
I just want to mention some work we did last August [2002] on how well these multiplexes work on actual forensic case samples because, as you know, we're a research and development lab. We build new things, but we don't actually run forensic cases in our lab. Butler: Slide 13
We took the multiplexes to the U.S. Army Criminal Investigation Laboratory (USACIL) in Atlanta. A graduate student named Rich Schoske, who was working in my lab, went down there. We tested on six cases that had been run previously. We examined those with the NIST 20plex, 9plex (which is just the minimal haplotype in the multiplex) 11plex, and 18plex and compared them with the Y–plex 5 and Y–plex 6 commercial kits.
I'm not going to go into great detail, but just to show on this particular case with the 20plex from USACIL, this is the differential extraction—the female fraction and the male fraction. Then, this is a followup with another analysis of the male fraction, the victim, and the suspect. We got all the loci to amplify just fine in these actual cases. This is from 1 nanogram of DNA, and these other little peaks are dye blobs that are present from impurities in the primers. But we don't get any amplification in the victim in the female fraction. There's a little bit of leakage because you don't have complete, clean differential extraction all the time. Butler: Slide 14
Just to show sensitivity with these assays, this is 50 picograms of male DNA, 32-cycle PCR (polymerase chain reaction), with our 11plex, and you can see a quite nice signal, over 2,000 RFUs (relative fluorescent units). I guess you can't see it on here, but it's on the paper that you have. Butler: Slide 15
In terms of specificity, how does it handle excess female DNA if you were to do it without a differential extraction? This is a 1:1,600 mixture, so 0.5 nanograms of male DNA, which is shown up here by itself, and 800 nanograms of female are mixed in. You can see the same profile between the mixture and the actual sample by itself. There are a few nonspecific peaks which you see here, but they fall outside the expected allele ranges. Therefore, you can tighten them just fine with a genotyper macro with this system. Butler: Slide 16
As I mentioned, we've been working on population typing for the Y chromosome. This is 647 males that have been run. We typed them first with Identifiler to be able to verify that all the samples are unique and are actually males. Then we ran them with the 22 STRs, with our 20plex and 11plex. Several of these loci overlap each other. Butler: Slide 17
They've also been typed with 50 Y–SNPs. Pete Vallone will be talking more about this work on Wednesday. And we've also typed them with a kit that is commercially available from Marligen. We've typed these with mini-STRs in collaboration with Bruce McCord, and we'll hear more about that tomorrow [Tuesday]. Then you heard this morning from Sandy Calloway about the work with typing these exact same samples with the Roche mitostrips for mitochondrial HVI and HVII.
So these are the multiplexes that we typed through high-throughput typing. In about 100 hours of typing, you can actually go through these 650 samples and get all these loci typed—that's about a week's worth of work on a 3100. Twenty-seven PCR products and 22 different loci are typed, and we generated 17,388 allele calls with these two multiplexes. So with the ability to multiplex, you can type things very, very quickly, allowing you to get access to this genetic data and understand what's going on in terms of population variation. Butler: Slide 18
I'll finish up by talking about the work we've done to make a standard reference material to guarantee that when you type samples in your laboratory they'll be consistent with other laboratories and databases that will be developed in this area.
This is our SRM. We have five male samples pictured here labeled A, B, C, D, and E, and F is a female. The female sample can serve as a negative control for male-specific assays. Butler: Slide 19
There's 100 nanograms of each component, so it's about 2 nanograms per microliter or 50 microliters in all. We have completely sequenced 22 loci to verify the number of repeats. Nine additional loci have been typed, and 42 Y–SNPs have been typed of all these samples. You can go to this Web site at NIST (i.e., the SRM Web site) and obtain this SRM. It can be used to verify results with any primer sets because we've sequenced the repeat region as well as the flanking regions. This will help U.S. laboratories meet the DNA Advisory Board (DAB)-FBI standards. Butler: Slide 20
How well do we know the results from each of these markers? This is the number of overlapping results at each of these markers. You can see this is the typing we did in our lab, the sequencing, the typing, and the number of different multiplexes or different tests that were run to get results for that locus. Then we also did an interlaboratory study with these same samples. We sent samples to ReliaGene, to OligiTrail, and over to Peter de Knijff's lab in The Netherlands, where sequencing was performed on the loci as indicated here. So we know very well what the results are on these. Butler: Slide 21
These are our laboratory's results from Y–plex 6. There's no result on the female DNA, and these are the types on the others. Butler: Slide 22
I just want to point out that, if you use this SRM with the current macro for Y–plex 6, this is an allele 20 at DYS385, and it's outside the bin for the 385 macro. So if you were to call that, it would show up only as a 1717 instead of a 1720 as it's supposed to be. You have to be careful with using the macro as it exists now, making sure that a peak is actually there. Butler: Slide 23
This is with the Promega kit, which has more alleles and an allelic ladder and a wider range. The 1720 is called correctly here. These were done with the Promega's run in our lab, the Promega prototype kit, and the SRM components, so all the types agreed with what we sequenced. Butler: Slide 24
Also, this is the 20plex against the same samples. All the loci are typed here. Butler: Slide 25
I'm just going to illustrate the sequences for those, just to show you how we sequenced them. Here are the 13, 11, 11, 11, and 12 TAT repeats. All these loci, which include all the U.S. haplotype, have been fully sequenced so that we know the exact repeat structure. Butler: Slide 26
In terms of dealing with ones that have multiple bands, you have to run a gel to separate those out, cut them out, and then you can sequence each of those bands separately. That agrees with the Y–plex 6 result here of a 12 and a 15 with the 2 sequences that we saw. Butler: Slide 27
So as I mentioned, we sent these out for interlaboratory confirmation to ReliaGene, who typed them with their two kits with the loci listed here, to OligiTrail—you'll hear from Debang Liu in a little while about the work that they've been doing with locus-specific brackets (LSBs)—and to Peter de Knijff in the Netherlands, who sequenced the loci that are shown here. Butler: Slide 28
Here are some results from Debang's OligiTrail typing. These are the LSBs, and we get the correct types using the LSBs. Butler: Slide 29
I will just briefly mention the work we've done with the Y–SNPs. As I mentioned, Pete Vallone on Wednesday morning will go into more detail about this. We developed methods that work on ABI (Applied Biosystems) 310 and 3100 platforms, and we also have typed with the Luminex platform. This is the primer extension; that is, the same as what Tom Parsons talked about with the mitochondrial DNA assay that Pete Vallone developed. We also have assays that are multiplexed at both the PCR level and at the SNP detection level for Y–SNP detection. The G on the top is a blue peak versus a T on the bottom being a red peak, so it's very simple to type with this system. Butler: Slide 30
Allele-specific hybridization is how the Marligen kit works. Here, you're just looking at the variation in terms of which peak is higher based on its relative fluorescence between the two alleles that you're preparing. As I mentioned, we've typed these in the 42 Y–SNPs that are present in the Marligen kit. Therefore, all commercially available kits at the moment have been typed on SRM 2395. You can use them to verify how your lab is performing once you get into Y-chromosome testing. Butler: Slide 31
So here are the results of some of these Y–SNPs. You can see every single example comes with a different pathway and lineage, so it makes it very nice to be able to distinguish different haplogroups on the Y chromosome as well.
This is the certificate. As you can imagine, NIJ has helped quite a bit in the funding of producing this SRM, so they're mentioned on the certificate. You can go to that Web site and get more information on SRM 2395. Butler: Slide 32
We also have information on STRBase about this, including publications on Y–STRs and Y–SNPs, allelic information on more than 20 loci, downloadable PowerPoint presentations on STRs from the Y chromosome and Y–SNPs, links to other sites, and information on newly available assays. So you can download the reprint of that paper I showed you with the 20plex just by going to our Web site. Then the Y–STRs have been mapped along the chromosome, and you can see where they are relative to each other. Butler: Slide 33
This is an illustration of a fact sheet from STRBase. It is kind of cut off here, but this is a DYS392 locus. We include all the primer sets that have been published, as well as our own multiplex primers; and we have all the alleles that have been seen, hot links to databases of extremely rare alleles, and references to publication numbers based on when those alleles were reported. Butler: Slide 34
So we'd like to collect varying alleles on Y–STRs, just like we're collecting them on the autosomal STRs. So as you discover these, please send us that information. We'll catalogue it within STRBase.
Allele nomenclature is very important when it comes to Y–STRs. This has caused problems in proficiency tests in the past and it's important to realize that there are different ways that laboratories in the literature have called some of these STRs, based on which parts of the repeat are variable versus the total repeats (i.e., the invariant plus the variable parts). Butler: Slide 35
This is the accepted nomenclature now for DYS19, which is 10 to 19 repeats. It includes an invariant 3 and then a variable 7 to 16. DYS439, which is now part of the core loci, has had three different methods for nomenclatures published in the literature and this is the one that's now been widely accepted, which is only the variable one. This is important to keep in mind as you're looking at this.
The big benefit, at least I think, of this SRM is that it will serve as an international standard to help resolve some of these issues with nomenclature.
I just want to summarize the work that we've done. We've developed new assays, the 20plex or the manly-plex. We've evaluated typing methodologies and developed new assays for Y–SNPs. We've created a standard reference material and have put all that information on STRBase to make it available to everyone. We've now generated more than 30,000 allele calls in the Y chromosome, which helps us understand which optimal markers to use in U.S. populations. Butler: Slide 36
These are our current publications, and we have several others planned and ready to go. Butler: Slide 37
I want to acknowledge gratefully the funding we've received from NIJ and NIST's Office of Law Enforcement Standards that allows us to be able to do this work. Butler: Slide 38
This is my project team: Pete Vallone, Margaret Kline, and Dave Duewer; and Rich Schoske just graduated from American University about a month and a half ago and is now working at Walter Reed Army Medical Center .
I'd also like to thank the wonderful collaborators that we've had on these projects: Mike Hammer and Alan Redd. You'll hear from Alan on Wednesday on the work he has been doing on the Y chromosome. Dave Carlson from Marligen helped with some of the Y–SNP typing information, and Del Price and Clem Smetana at USACIL helped with the casework samples.
Thank you very much for your attention.
MS. TOMSEY: Thank you, John.