1. FidoTyping TM and FidoSearch TM : Validation of a forensic canine mitochondrial DNA protocol and a new on-line canid mitochondrial hypervariable region database Terry Melton, PhD, Janusz Sikora, PhD, Verónica Fernandes, MSc, and Luísa Pereira, PhD Mitotyping Technologies, State College, PA, USA, and Institute of Pathology and Molecular Immunology, Porto, Portugal www.mitotyping.comwww.mitotyping.com or twm107@mitotyping.com

2. DISCLOSURE: Yes, we are… NOTE: Trademarks are names only; no proprietary methods are used; database and search software will be freely available for use on the Mitotyping website at www.mitotyping.com

3. Dog hairs: frequent evidence  Usually transfer evidence: suspect to crime scene or victim victim to suspect cases where dogs actually involved in crime  Microscopy can usually distinguish dog from human hair:  Dog hair is a good source of mtDNA and nuclear DNA CanineHuman

4. mtDNA analysis of dog hair  Success with old and small human hairs means dog hair evidence analysis logical extension of skill set  Hair microscopy useful adjunct to DNA analysis, especially in dogs  Dog hairs have much more DNA on average than human hairs  Protocols are exactly the same for dog hairs  Differences are in primer pairs that amplify canine mtDNA control region

5. Canid mtDNA: ~ 16,800 bp Two hypervariable regions: 15431-16092 16450-00014 A 10 bp repeat region from about 16,130-16430 (not forensically informative) Forensically informative regions: 10 bp repeats Canine mtDNA is very similar to human mtDNA

6. Validation:Canine mtDNA Analysis  This study was a “material modification” of human protocols  Three primer pairs amplify three products ~300-350 bp  18 dogs and wolves analyzed  Undercoat and guard hairs amplified equally and hairs ≤0.5 cm worked well  Used Kim et al. 1998 reference dog as a benchmark to record polymorphisms, like the rCRS for humans  Some species cross-hybridization was observed for certain primers; in future all amplification products will be sequenced and searched in GenBank to identify species if not apparently canid.

7. Dog mtDNA population genetics  Allard et al. JFS 54:289 (2009): study of 552 control regions: No genetic basis for grouping dogs either by breed or geographic region A single database comprised of purebred and mixed dogs is suitable for the US Four main haplogroups: A-D 53% of dogs had one of nine haplotypes The remaining 134 haplotypes are low frequency (<2%) However, the most common haplotype, B1a, N=59, has dogs from 38 breeds represented. Question: Can hair microscopy be used to aid a case in association with a particular breed?

8. Distribution of haplotypes Courtesy of Webb and Allard, JFS 54:289-304 (2009) N=552 The most common types are in haplogroups A and B Many more rarer types Other publications with sizeable datasets show similar distributions and same common types. [Savolainen/Vila/Gundry] D C B A

9. Oops…PREPRINT coming…

10. Are regional differences are statistically significant?  May be largely accounted for by the common types…  However, common types for forensic mtDNA analysis are already problematic and somewhat useless  Analysis needs to be done for regional differences in rare types (that is: Is a rare type in one area common in another area?)

11. Caveats?  Purebred dog lineages may have substructure  For example, golden retrievers and labrador retrievers are highly inbred  In Allard’s paper, 18/44 labradors shared haplotype A16a, [but the remaining dogs had 11 other types…]  14/39 goldens shared A33c, 10 had B1a, 5 had A16a, [but the remaining others had 10 other types…]  A local database may be needed when a particular case involves possible inbreeding  Dogs are much less diverse than humans; stats are even less powerful  An adjunct method in a case with abundant evidence may be the best use. All the data are not in.

12. Impetus for FidoSearch:

13. GenBank:A wealth of canid data  Pereira and Fernandes extracted all canid mtDNA data  Canis familiaris (domestic dog) plus wolf, dingo, and coyote  N=3,947  Mostly control region sequences (full and partial) and some coding regions

14. Distribution ContinentDogsWolvesAll Africa35237 Asia8471681015 Australia2130 Europe13204371757 NorthAmerica84565910 SouthAmerica13215 Total32736743947

15. FIDO?

16. FidoSearch:  Software package and database for searching canine mtDNA sequences  Similar to Mitosearch (FBI human)  Free to public/online at www.mitotyping.com www.mitotyping.com  Database and software can be used & examined but not downloaded  All sequences are identified by GenBank Accession Number

17. Database  FidoSearch unique identifier for sequence as it was downloaded from GenBank  GenBank Accession Number  Species name (C. familiaris, C. lupus, etc)  Breed name if available  Continent of origin  Range of sequence data in mtDNA molecule  Polymorphisms based on a standard reference dog from Kim et al. 1998  Bibliographic references/Authors

18. And:  Whole canid genomes: N=108  Control regions: N=3,816  Total searched for forensics? 3,924  Searches are always done for partial as well as full profiles…  Coding regions: N=23 (goal? Add SNPs)

19. Software Interface

20. Output: Part 1: The profile and parameters set

21. Part 2: The total sequences searched and subsets

22. Part 3: Number of matches and frequency

23. Part 4: Sequences of matched samples, including range, continent, species

24. Sam I Am Melton  High frequency type  Observed 396 times in 3847 dogs in database  Upper bound frequency: 11.55%

25. Conclusions  For known dog samples (especially hairs), mtDNA analysis can add another dimension of information to your crime scene evidence  Exclusions highly useful  Failures to exclude: problematic for certain breeds when hair microscopy cannot help corroborate  May be highly useful for rare types based on current literature  The FidoSearch Database and Software provide contextual meaning to a match: a 95% upper bound confidence limit can be applied  The system itself (typing and searching) is supported by scientific literature for courtroom presentation

27. My invitation to dog DNA fans?  Mitotyping will provide FidoSearch free to any group that contributes their dog mtDNA sequences to the database  We will periodically update the software and database for all these contributors when more sequences are added  We estimate that there are an additional >4000 dog profiles in the possession of individual research groups  This effort would aid in developing regional data sets for the whole forensic community  Contact me at twm107@mitotyping.com for more infotwm107@mitotyping.com