Forensic characterization and statistical considerations of the CaDNAP 13-STR panel in 1,184 domestic dogs from Germany, Austria, and Switzerland.

Crime scene samples originating from domestic dogs such as hair, blood, or saliva can be probative as possible transfer evidence in human crime and in dog attack cases. In the majority of such cases canine DNA identification using short tandem repeat (STR) analysis is the method of choice, which demands, among others, a systematic survey of allele frequency data in the relevant dog populations. A set of 13 highly polymorphic canine STR markers was used to analyze samples of 1,184 dogs (including 967 purebred dogs) from the so-called DACH countries (Germany, Austria, Switzerland). This CaDNAP 13-STR panel has previously been validated for canine identification in a forensic context. Here, we present robust estimates of allele frequencies, which are essential to assess the weight of the evidence by estimating the probability of a matching DNA profile within the dog population under question, e.g. in the form of a random match probability (RMP). The geographical provenance of the tested dogs showed a negligible influence on the observed genotype variation. Therefore, we combined the STR data from all three countries into a single dog population sample (DPS). In contrast, pronounced genetic differentiation between dog breeds was found by principal component analysis and sub-structure analysis with the STRUCTURE software. These findings entailed the need to account for the effects of DPS breed composition on allele frequency estimates. A possible strategy, which was favored here, relies on collecting a DPS that is guided by the breed composition of the relevant dog population. In total, dogs from 166 different breeds were included in our DPS, 64 of them including at least 5 individuals (n = 771 dogs). Sampling reflected the abundance of breeds in the DACH countries with the following being the most common ones: German Shepherds (population frequency: 14.3%), Dachshunds (5.9%), Labrador Retrievers (3.9%), and Golden Retrievers (3.2%). The pedigree listing of the purebred dogs in our DPS ranked German Shepherds (DPS frequency 8.5%) first, followed by Labrador Retrievers (3.9%), Golden Retrievers (3%), and Dachshunds (2.5%). RMP values based on overall allele frequencies and accounting for substructure using FST between breeds ranged between 10-13 and 10-14 and represent a conservative approach of RMP assessment.

Dog breed affiliation with a forensically validated canine STR set.

We tested a panel of 13 highly polymorphic canine short tandem repeat (STR) markers for dog breed assignment using 392 dog samples from the 23 most popular breeds in Austria, Germany, and Switzerland. This STR panel had originally been selected for canine identification. The dog breeds sampled in this study featured a population frequency ≥1% and accounted for nearly 57% of the entire pedigree dog population in these three countries. Breed selection was based on a survey comprising records for nearly 1.9 million purebred dogs belonging to more than 500 different breeds. To derive breed membership from STR genotypes, a range of algorithms were used. These methods included discriminant analysis of principal components (DAPC), STRUCTURE, GeneClass2, and the adegenet package for R. STRUCTURE analyses suggested 21 distinct genetic clusters. Differentiation between most breeds was clearly discernable. Fourteen of 23 breeds (61%) exhibited maximum mean cluster membership proportions of more than 0.70 with a highest value of 0.90 found for Cavalier King Charles Spaniels. Dogs of only 6 breeds (26%) failed to consistently show only one major cluster. The DAPC method yielded the best assignment results in all 23 declared breeds with 97.5% assignment success. The frequency-based assignment test also provided a high success rate of 87%. These results indicate the potential viability of dog breed prediction using a well-established and sensitive set of 13 canine STR markers intended for forensic routine use.

Isolation and whole genome analysis of endospore-forming bacteria from heroin.

Infections caused by endospore-forming bacteria have been associated with severe illness and death among persons who inject drugs. Analysis of the bacteria residing in heroin has thus been biased towards species that affect human health. Similarly, exploration of the bacterial diversity of seized street market heroin correlated with the skin microflora of recreational heroin users insofar as different Staphylococus spp. or typical environmental endospore formers including Bacillus cereus and other Bacilli outside the B. cereus sensu lato group as well as diverse Clostridia were identified. In this work 82 samples of non-street market ("wholesale") heroin originating from the German Federal Criminal Police Office's heroin analysis program seized during the period between 2009 and 2014 were analyzed for contaminating bacteria. Without contact with the end user and with only little contaminations introduced by final processing, adulteration and cutting this heroin likely harbors original microbiota from the drug's original source or trafficking route. We found this drug to be only sparsely populated with retrievable heterotrophic, aerobic bacteria. In total, 68 isolates were retrieved from 49 out of 82 samples analyzed (60% culture positive). All isolates were endospore-forming, Gram-positive Bacilli. Completely absent were non-endospore-formers or Gram-negatives. The three most predominant species were Bacillus clausii, Bacillus (para)licheniformis, and Terribacillus saccharophilus. Whole genome sequencing of these 68 isolates was performed using Illumina technology. Sequence data sets were assembled and annotated using an automated bioinformatics pipeline. Average nucleotide identity (ANI) values were calculated for all draft genomes and all close to identical genomes (ANI>99.5%) were compared to the forensic data of the seized drug, showing positive correlations that strongly warrant further research on this subject.

Technical Note: Simple, scalable, and sensitive protocol for retrieving Bacillus anthracis (and other live bacteria) from heroin.

We describe a culture-based method suitable for isolating Bacillus anthracis and other live bacteria from heroin. This protocol was developed as a consequence of the bioforensic need to retrieve bacteria from batches of the drug associated with cases of injectional anthrax among heroin-consumers in Europe. This uncommon manifestation of infection with the notorious pathogen B. anthracis has resulted in 26 deaths between the years 2000 to 2013. Thus far, no life disease agent has been isolated from heroin during forensic investigations surrounding these incidences. Because of the conjectured very small number of disease-causing endospores in the contaminated drug it is likely that too few target sequences are available for molecular genetic analysis. Therefore, a direct culture-based approach was chosen here. Endospores of attenuated B. anthracis artificially spiked into heroin were successfully retrieved at 84-98% recovery rates using a wash solution consisting of 0.5% Tween 20 in water. Using this approach, 82 samples of un-cut heroin originating from the German Federal Criminal Police Office's heroin analysis program seized during the period between 2000 and 2014 were tested and found to be surprisingly poor in retrievable bacteria. Notably, while no B. anthracis was isolated from the drug batches, other bacteria were successfully cultured. The resulting methodical protocol is therefore suitable for analyzing un-cut heroin which can be anticipated to comprise the original microbiota from the drug's original source without interference from contaminations introduced by cutting.

A proposal for standardization in forensic canine DNA typing: allele nomenclature of six canine-specific STR loci.

In this study a proposal for the allele nomenclature of six polymorphic short tandem repeat (STR) loci (PEZ3, PEZ6, PEZ8, PEZ10, FHC2161, and FHC2328) for canine genotyping (Canis lupus familiaris) is presented. The nomenclature is based on the sequence data of the polymorphic region of the microsatellite markers as recommended by the DNA commission of the International Society of Forensic Haemogenetics (ISFH) in 1994 for human DNA typing. To cover commonly and rarely occurring alleles, a selection of homozygous and heterozygous animals were analyzed and subjected to sequence studies. The alleles consisted of simple tri- and tetra-nucleotide repeat patterns as well as compound and highly complex repeat patterns. Several alleles revealing the same fragment size but different repeat structures were found. The allele designation described here was adopted to the number of repeats, including all variable regions within the amplified fragment. In a second step the most commonly occurring alleles were added to an allelic ladder for each marker allowing a reliable typing of all alleles differing in size. A total number of 142 unrelated dogs from surrounding municipal animal homes, private households, and canines in police duty were analyzed. The data were added to a population database providing allele frequencies for each marker.