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The aim of this experiment was to measure enteric methane (CH4) emission and its relation with rumen digestion in dairy cows fed diets rich in 1 of the 2 carbohydrate sources, starch or sugar. The rations were based on late first-cut grass–clover silage supplemented with wheat (Wh), NaOH-treated wheat (Wh+NaOH), sugar beet molasses (Mo), or sugar beet molasses with addition of sodium bicarbonate (Mo+Bic). Wheat and molasses made up 35% of dry matter in the 2 diets with molasses and wheat, respectively. Four cows fitted with ruminal, duodenal, and ileal canulae were used in a 4 × 4 Latin square design. Nutrient digestibility was measured using chromium oxide and titanium oxide as flow markers, and emissions of CH4 and hydrogen were measured via open-circuit indirect calorimetry on 4 consecutive days. Data were analyzed using PROC MIXED of SAS (version 9.4; SAS Institute Inc., Cary, NC) with treatment and period as fixed effects and cow as random effect. Furthermore, orthogonal contrasts were calculated. The cows produced 32.5, 33.6, 36.2, and 35.1 L of CH4/kg of dry matter intake (DMI) on diets Wh, Wh+NaOH, Mo, and Mo+Bic, respectively. The emission of CH4 per day, per kilogram of DMI, and per kilogram of energy-corrected milk as well as daily hydrogen emission were higher on the Mo diet compared with the Wh diet. With the present inclusion of wheat and molasses in the diet, no effects of NaOH treatment of wheat or of sodium bicarbonate supplementation to the Mo diet could be demonstrated on CH4 emission expressed per kilogram of DMI or per kilogram of energy-corrected milk. The duodenal flow of starch was higher when wheat was treated with NaOH. Under the conditions in the present experiment, ruminal NDF digestibility was not affected by carbohydrate source, NaOH treatment of wheat, or bicarbonate supplementation. Total volatile fatty acid concentration in the rumen and the proportions of acetate and propionate were not affected by carbohydrate source, NaOH treatment of wheat, or bicarbonate supplementation. Likewise, we could not show any influence of diet on microbial protein synthesis or efficiency of microbial protein synthesis expressed as grams of microbial protein synthesis per kilogram of true rumen-digested organic matter. We concluded that CH4 emission was increased when wheat was replaced by molasses, whereas no effect of manipulating rumen fermentation by NaOH treatment of wheat or addition of bicarbonate to molasses could be found with a level of approximately 25% of dry matter from starch and sugar, respectively. [ABSTRACT FROM AUTHOR]

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation. [ABSTRACT FROM AUTHOR]

Abstract: Emerging next-generation sequencing technologies will enable DNA analyses to add pigmentation predictive and ancestry informative (AIM) SNPs to the range of markers detectable from a single PCR test. This prompted us to re-appraise current forensic and genomics AIM-SNPs and from the best sets, to identify the most divergent markers for a five population group differentiation of Africans, Europeans, East Asians, Native Americans and Oceanians by using our own online genome variation browsers. We prioritized careful balancing of population differentiation across the five group comparisons in order to minimize bias when estimating co-ancestry proportions in individuals with admixed ancestries. The differentiation of European from Middle East or South Asian ancestries was not chosen as a characteristic in order to concentrate on introducing Oceanian differentiation for the first time in a forensic AIM set. We describe a complete set of 128 AIM-SNPs that have near identical population-specific divergence across five continentally defined population groups. The full set can be systematically reduced in size, while preserving the most informative markers and the balance of population-specific divergence in at least four groups. We describe subsets of 88, 55, 28, 20 and 12 AIMs, enabling both new and existing SNP genotyping technologies to exploit the best markers identified for forensic ancestry analysis. [Copyright &y& Elsevier]

Non-uniform phenotyping of five case work samples were observed in the D12S391 locus. The samples were typed at least twice with the AmpFℓSTR® NGM SElect™ PCR Amplification Kit and different alleles were called with GeneMapper® ID-X in the different experiments. Detailed analyses of the electropherograms suggested that the individuals were heterozygous with two alleles that differed in size by one nucleotide. This was confirmed by amplifying the samples with the PowerPlex® ESX 17 system. D12S391 is a complex STR with variable numbers of AGAT and AGAC repeats. Second generation sequencing revealed that separation of two alleles differing by one nucleotide in length was poor if the number of AGAT repeats in the short allele was higher than in the long allele. A total of 45 individuals with microvariants or off-ladder alleles in D12S391 were sequenced. Thirty different alleles were detected and sixteen of these were not previously reported. [ABSTRACT FROM AUTHOR]

Sequenom launched the first commercial SNP typing kit for human identification, named the iPLEX® Sample ID Plus Panel. The kit amplifies 47 of the 52 SNPs in the SNPforID panel, amelogenin and two Y-chromosome SNPs in one multiplex PCR. The SNPs were analyzed by single base extension (SBE) and Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). In this study, we evaluated the accuracy and sensitivity of the iPLEX® Sample ID Plus Panel by comparing the typing results of the iPLEX® Sample ID Plus Panel with those obtained with our ISO 17025 accredited SNPforID assay. The average call rate for duplicate typing of any one SNPs in the panel was 90.0% when the mass spectra were analyzed automatically with the MassARRAY® TYPER 4.0 genotyping software in real time. Two reproducible inconsistencies were observed (error rate: 0.05%) at two different SNP loci. In addition, four inconsistencies were observed once. The optimal amount of template DNA in the PCR was ≥10ng. There was a relatively high risk of allele and locus drop-outs when ≤1ng template DNA was used. We developed an R script with a stringent set of “forensic analysis parameters” based on the peak height and the signal to noise data exported from the TYPER 4.0 software. With the forensic analysis parameters, all inconsistencies were eliminated in reactions with ≥10ng DNA. However, the average call rate decreased to 69.9%. The iPLEX® Sample ID Plus Panel was tested on 10 degraded samples from forensic case-work. Two samples could not be typed, presumably because the samples contained PCR and SBE inhibitors. The average call rate was generally lower for degraded DNA samples and the number of inconsistencies higher than for pristine DNA. However, none of the inconsistencies were reproduced and the highest match probability for the degraded samples typed with the panel was 1.7E−9 using the stringent forensic analysis parameters. Although the relatively low sensitivity of the iPLEX® Sample ID Plus Panel makes it inappropriate for typing of trace samples from crime scenes, the panel may be interesting for relationship testing and for identification of e.g. samples in biobanks because of the low reagent costs, the limited hands-on time of the iPLEX® assay and the automatic analysis of the mass spectra. [ABSTRACT FROM AUTHOR]

The objective was to study the effect of rumen-escape starch and coarseness of ingredients in pelleted concentrates on performance, carcass quality and rumen wall characteristics in rosé veal calf production. Two alternative concentrates (Coarse and Slow) were compared with a traditional (Control) concentrate. Control was based on finely ground ingredients, whereas in Coarse, the same ingredients were coarsely ground resulting in a mean particle size before pelleting of 1.5 in Coarse and 0.6 mm in Control. Slow compared with Control and Coarse contained finely ground sorghum and corn instead of barley and wheat which increased the amount of rumen-escape starch to 59 compared with 22 g/kg in Control and Coarse. All concentrates had the same total starch (362 g/kg), NDF (168 g/kg), CP (154 g/kg) and DE (15.5 MJ/kg DM) content and a pellet diameter of 3.5 to 4 mm. Use of an ‘indicator of starch digestibility’ method gave a value of 98.6% for Control and Coarse and 91.1% for Slow (P < 0.001). A total of 57 Holstein bull calves (n = 19 per treatment) were offered one of the three concentrates ad libitum from weaning ( $$$2\frac{1}{2}$$$ months of age) to slaughter (<10 months of age). Concentrate intake was recorded individually. Barley straw was available ad libitum but intake was not recorded. Average daily gain (1.43 kg/day), concentrate conversion efficiency (3.7 kg DM concentrate/kg gain), LW at slaughter (386 kg), carcass weight (194 kg) and EUROP conformation (3.9) were not affected by type of concentrate (P > 0.05). Papillae length and shape evaluated in atrium ruminis and the cranial part of the ventral rumen sac at slaughter were not affected by type of concentrate (P > 0.05). Rumen wall characteristics showed degrees of plaque formation (i.e., papillary aggregation), hyperaemia and necrotic areas in all treatment groups, but with no general difference between type of concentrate (P > 0.05). Incidence of liver abscesses (LAs, 16%) was not affected by type of concentrate (P > 0.05). There were no differences in performance or rumen wall characteristics between liver-abscessed and non-abscessed calves. The results show a high level of production performance with the three types of pelleted concentrates and indicates that neither the more coarse ingredients nor the additional rumen-escape starch tested, when fed ad libitum, could improve rumen wall characteristics or reduce LAs of rosé veal calves. [ABSTRACT FROM PUBLISHER]

Abstract: The GenPlex™ HID System (Applied Biosystems – AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250–500pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power. [Copyright &y& Elsevier]

Single nucleotide polymorphism genotyping provides a supplement for conventional short tandem repeats-based kits currently used for human identification. GenPlex (Applied Biosystems (AB), Foster City, CA) is an SNP-genotyping kit based on a multiplex of 48 informative, autosomal SNPs from the SNPforID Consortium. Our objective was to setup, implement, and validate a small and affordable automated liquid-handling robot for forensic casework samples (buccal swaps on FTA-paper and Qiagen purified blood). The reaction scheme consisted of numerous steps and was cumbersome to perform consistently manually. Automation was accomplished with a Biomek-3000 (Beckmann Coulter) laboratory-automated workstation using five in-house-developed methods. All methods allowed the user to select the number of subsequent injections to the capillary electrophoresis instrument (ABI 3130xl, AB) enabling processing of both partial and full plates. A total of 286 samples were analyzed in duplicates with the GenPlex reaction using the Biomek-3000. The results were compared with those obtained from the same samples using the SNaPshot(AB) single-base extension system. Full concordance of the results was obtained in all but one sample. The results demonstrate that the Biomek-3000 can perform a series of complex reactions leading to highly consistent forensic genetic SNP-typing results. [ABSTRACT FROM AUTHOR]

Abstract: We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics. [Copyright &y& Elsevier]

Abstract: A total number of 149 individuals from Iran (Persians, Lurs and Kurds) were analyzed for 49 autosomal SNPs using PCR, SBE and capillary electrophoresis. No deviation from Hardy–Weinberg expectations was observed. One SNP pair (rs1015250–rs251934) showed significant linkage disequilibrium in Kurds. However, this was most likely due to chance. High intrapopulation variability and no significant population structure were observed among the three ethnic groups from Iran. Pairwise F ST values obtained from the mean numbers of pairwise differences between SNP profiles were calculated for Persians, Lurs, Kurds and eighteen other worldwide populations. For each of the three Iranian ethnic groups, the lowest F ST values calculated between an Iranian and non-Iranian populations were observed between Iranians and populations in Iraq and Turkey. The three Iranian ethnic groups grouped together with other West Asian populations in the MDS plot drawn from the F ST values. Statistical parameters of forensic interest calculated for the Iranian ethnic groups showed values of the same order of magnitudes as those obtained for Asians. The mean match probability calculated for the 49 SNPs ranged from 1.7x10−18 for Kurds to 1.3x10−19 for Persians. Despite the low level of genetic structure observed among Persians, Lurs and Kurds, a single autosomal SNP database should be used with care when extending its forensic application to other Iranian ethnic groups. [Copyright &y& Elsevier]

Abstract: X-chromosome markers may complement the results obtained from other genetic markers in complex relationship cases. Until now, reports on relationship testing using X-chromosome markers have mainly included data of short tandem repeats (STRs) while little data on single nucleotide polymorphisms (SNPs) in relationship testing have been published. We selected 25 highly polymorphic biallelic SNPs distributed through the human X-chromosome. One 25-plex PCR reaction and one 25-plex single base extension (SNaPshot) reaction were developed. The maximum size of the PCR products was 120bp and the size of the SBE primers varied between 18 and 85 nucleotides. We analyzed the allele and haplotype frequencies in 1078 unrelated males. All the SNPs were polymorphic and the lowest minor allele frequency was 0.103. All the haplotypes were unique. The forensic parameters were calculated in the Danish and Somali populations. In the Danish population (N =93), the power of discrimination (PD) in females was one in 4.4×109 individuals and the PD in males was one in 2.6×106. The PD in Somalis (N =91) was one in 2.7×109 in females and one in 1.7×106 in males. Finally, we present an example of how the 25 X-chromosome SNP-multiplex may help to solve a complex immigration case. [Copyright &y& Elsevier]

• Uyghur and Kazakh populations from Xinjiang, China were investigated with the Precision ID Ancestry Panel that amplifies 165 ancestry-informative SNPs. • STRUCTURE and PCA analyses showed that the Precision ID Ancestry Panel could not differentiate Uyghur and Kazakh populations. However, these populations were separated from Europeans, Middle Easterners, South/Central Asians, and East Asians. • With the introduction of Uyghur and Kazakh reference populations in GenoGeographer, ancestry inference of individuals from these ethnic groups reached likelihood ratios ≥106. Autosomal ancestry informative markers (AIMs) are important markers for inferring ancestry of humans. In the present study, we typed 105 Uyghurs and 94 Kazakhs with the Precision ID Ancestry Panel that amplifies 165 autosomal AIMs. No statistically significant deviation from Hardy-Weinberg equilibrium and no linkage disequilibrium between loci was observed after Bonferroni correction. STRUCTURE and PCA analyses showed that Uyghurs and Kazakhs appeared as admixed individuals of primarily European and East Asian ancestry and were clearly differentiated from Europeans, Middle Easterners, South/Central Asians, and East Asians. However, it was not possible to differentiate the two populations from each other and they were also difficult to differentiate from Greenlanders, a population with European/Inuit admixture. GenoGeographer was used to evaluate the weight of the evidence. Initially, the results showed that the majority of AIM profiles from Uyghur and Kazakh individuals were not represented by any of the 36 reference populations of the GenoGeographer database. Consequently, it was not reasonable to infer the ancestry of these individuals. A randomly selected subset of the studied populations (75 Uyghur and 75 Kazakh individuals) was used to construct two new reference populations for GenoGeographer, and ancestry prediction was performed on the remaining test individuals. A total of 42 out of 49 test individuals were represented by at least one population after the introduction of Uyghur and Kazakh reference populations. Likelihood ratios ≥106 were obtained when the alternative hypothesis was that the individual belonged to the South/Central Asian, East Asian, Middle Eastern, European, or the admixed Greenlandic population. [ABSTRACT FROM AUTHOR]

We have developed a PCR-based assay with co-amplification of 25 DNA fragments and detection of 35 Y chromosome SNPs with the SNaPshot technique. The Y SNP package can define 34 Y chromosome haplogroups and it can identify the majority of the Y chromosome haplogroups of interest in the populations relevant to forensic genetics in Denmark. We typed 194 Danes, 215 Greenlanders, and 201 Somalis, all males. A total of 21 different haplogroups were identified. In Danes, 11 haplogroups with frequencies from 0.5% to 38% were identified and three of the haplogroups, I, R1b*(xR1b1, R1b6, R1b8) and R1a1*(xR1a1b), were found in ∼91% of the population. In Greenlanders, 10 haplogroups with frequencies from 0.5% to 50% were identified and the haplogroups P*(xQ3a, R1), R1b*(xR1b1, R1b6, R1b8) and I, were found in ∼86% of the population. In Somalis, 14 haplogroups with frequencies from 0.5% to 79% were identified and the haplogroups E3b1*(xE3b1b) and K*(xN3, O, P) were found in ∼88% of the population. The distribution of haplogroups was compared to the distribution found in 65 males from West Africa. [Copyright &y& Elsevier]

Abstract: We report the results of an inter-laboratory exercise on typing autosomal single nucleotide polymorphisms (SNPs) with the GenPlex™ HID system (Applied Biosystems—AB). A total of 12 European and US forensic genetic laboratories typed 10 reference DNA samples and 3 samples with partly degraded DNA that only gave strong reactions with amplicons shorter than approximately 135bp and weak reactions up to 185bp. The samples and the critical reagents were sent to the participating laboratories from Copenhagen. In addition, the laboratories typed the three degraded DNA samples with one or more STR kits. When 500pg degraded DNA was used, the success rates of SNP typing in the laboratories varied from 15.6% to 98.6% (median: 94.6%). Only 1.6% of the unsuccessful SNP types were due to wrong SNP calling, while the remaining 98.4% were due to lack of amplification and uncertain results. Two laboratories counted for more than two-thirds of the non-successful results while seven laboratories reported no wrong SNP types. Two laboratories typed the degraded DNA samples successfully with the MiniFiler kit, while other commonly used kits gave incomplete STR profiles in all laboratories. Titration of the degraded DNA showed that the SNP typing results became less reliable with 100pg DNA and less. The median success rate of SNP typing of 10 reference samples with 50pg reference DNA was 86% (range: 44.5–99.2% among laboratories). The success rate of SNP typing of the reference samples decreased with smaller amounts of DNA. SNP typing with the GenPlex™ HID system provided more information from degraded DNA samples than any of the commercially available kits used by the participating laboratories. [Copyright &y& Elsevier]

Abstract: In certain crime cases, information about a perpetrator's phenotype, including eye colour, may be a valuable tool if no DNA profile of any suspect or individual in the DNA database matches the DNA profile found at the crime scene. Often, the available DNA material is sparse and allelic drop-out of true alleles is possible. As part of the validation of the IrisPlex assay in our ISO17025 accredited, forensic genetic laboratory, we estimated the probability of drop-out of specific SNP alleles using 29 and 30 PCR cycles and 25, 50 and 100 Single Base Extension (SBE) cycles. We observed no drop-out when the amount of DNA was greater than 125pg for 29 cycles of PCR and greater than 62pg for 30 cycles of PCR. With the use of a logistic regression model, we estimated the allele specific probability of drop-out in heterozygote systems based on the signal strength of the observed allele. The probability of drop-out in heterozygote systems depended on the signal strength of the observed allele as well as the number of PCR and SBE cycles. [Copyright &y& Elsevier]

Abstract: The Greenlandic population is characterised by migrations from two major areas: the northern parts of North America and Scandinavia. Y-chromosomal haplogroup (Y-HG) P*(xR1-M173)-M45 was previously found in up to 50% of Greenlanders. The aim of this study was to investigate the distribution of sub-HGs within Q-M242 in the Greenlandic population. A multiplex of 15 Y-chromosomal single nucleotide polymorphisms (Y-SNPs) covering major sub-HGs within Q-M242 was constructed using the AssayDesign software (Sequenom) and analysed on the Sequenom platform. Two sub-HGs within Q-M242 were found in the Greenlandic population, Q-L56, L57(xM19, M323, L529) and Q-NWT01(xM265). An increase of Inuit Y-HGs along the coastline from Qeqqata (30%) in the West to East Sermersooq (82%) was found. A corresponding decrease was found for European Y-HGs, from 59% in Qeqqata to 16% in East Sermersooq. [Copyright &y& Elsevier]

Abstract: Ancestry informative markers (AIMs) can be used in forensic genetics to infer the biogeographic ancestry of a person, e.g. from a DNA trace sample found at a crime scene. The European Forensic Genetics Network of Excellence (EUROFORGEN-NOE) selected a panel of more than 200 AIM SNPs with the purpose of developing multiplex assays for forensic ancestry testing. The SNPs were selected to discriminate between five major population groups (Africans, Europeans, Asians, Native Americans and Pacific Islanders). Here, we describe the development of five PCR multiplexes for detection with the Sequenom MassARRAY® system of 115 AIM SNPs from the EUROFORGEN selection panel. The five multiplexes allowed typing of 25, 33, 24, 13 and 29 AIM SNPs, respectively. Furthermore, one X and two Y chromosome SNP were included for sex determination. We evaluated the sensitivity and overall performance of each multiplex in a Danish population. The SNP call rates were 93.1%, 96.1%, 97.3%, 97.2%, and 97.1% for the five multiplexes, respectively. The typing results were robust when more than 1.25ng DNA was used in the PCR. The data analysis was performed using a custom made script in the statistical software R. This script called the phenotypes automatically by using information on peak heights, signal to noise ratios and allelic balances. Three SNPs were sequenced to elucidate skewed allelic balances. These results as well as the allelic frequencies for all SNPs will be presented. [Copyright &y& Elsevier]

Abstract: Brown and blue eye colours are primarily explained by the single nucleotide polymorphism (SNP) HERC2 rs12913832. However, the genetics of eye colours that appear to be neither blue nor brown are not well understood. In this study, 230 unrelated Italian individuals were typed for 32 SNP loci in pigmentary genes. High resolution digital images of the participants’ eyes were taken and the iris region was successfully extracted with the use of the custom designed software Digital Iris Analysis Tool (DIAT) from 218 of the 230 (95%) images. The software counted the numbers of blue and brown pixels in the iris region and calculated a Pixel Index of the Eye (PIE-score) that described the eye colours quantitatively. The PIE-score ranged from −1 to 1 (brown to blue). We investigated the association of the PIE-scores extracted from the eye images with the genotypes of the 32 pigmentary SNPs. We observed a statistically significant association between the PIE-scores and the SNP loci rs12913832, rs4778241, rs7495174 in the HERC2/OCA2 region and the locus rs16891982 in SLC45A2. [Copyright &y& Elsevier]

Abstract: Haploblocks are segments of the genome with little recombination that may be of interest in forensic and population genetics. Criteria to select autosomal haploblocks have been previously described, leading to the identification of candidate regions that, a priori, met the conditions to be used as forensic genetic markers. Still, the potential of X-chromosomal haploblocks remains unexplored. The present work aimed to provide basis for designing strategies for selection of X-haploblocks defined by single nucleotide polymorphisms (SNPs) using next generation sequencing approach. The potential application in population genetics and forensic studies was addressed. One of the conditions considered in the haploblock selection was the simultaneous inclusion of short tandem repeats (STRs) currently used in forensic casework to allow the distinction between SNP-defined haplotypes and increase the resolution for fine-scale studies. Given the size of the X chromosome (∼150Mbps), only four haploblocks could be selected in order to guarantee their independence. [Copyright &y& Elsevier]

Abstract: An assay was designed for typing 29 of the 52 SNPs in the SNPforID multiplex on the Sequenom® MassARRAY® analyzer 4 system. The dedicated TYPER 4 software was used for automatic allele calling. The sensitivity of the assay was tested using 0.5–40ng pristine template DNA in the PCR reaction. Overall, no difference was observed in the sensitivity, measured as call rate, for the different amounts of template DNA. Typing of paternity case work samples resulted in lower call rates (54%) as compared to the sensitivity test (89%), and more incorrect calls, 0.4% as compared to 0.1%. Finally, the ability of the software to detect 1:1, 1:3 and 1:5 DNA mixtures was investigated. Compared to the sensitivity test, the call rates of the 1:1 and 1:3 mixtures were low (64–74%), whereas the call rates of the 1:5 mixtures were similar (88–90%). However, in comparison with the case work samples, the call rates of the mixtures were not unusual and mixtures may be overlooked. [Copyright &y& Elsevier]

Abstract: A total of 249 FTA® card samples were investigated with the Identifiler® Direct kit. PCR amplification was performed according to the manufacturer''s protocol with modifications in the reaction volume (10–25μL), the number of PCR cycles (25–27) and the number of FTA-card punches (1–2). Full concordance was observed between the results obtained with the Identifiler® Direct kit and the washed punches typed with the Identifiler® kit. The success rate with one 1.2mm punch in a 15μL PCR reaction volume and 25 PCR-cycles was 93.3%. After retyping of the failed samples, the combined success rate was 98.7%. Overall, the quality of the results was reduced when the number of punches was increased or the PCR reaction volume was decreased. The Identifiler® Direct master mix may be used in combination with other primer sets for direct amplification of other loci, e.g. the 49 SNPs using the SNPforID multiplex assay. [Copyright &y& Elsevier]

Abstract: In this study, 33 single nucleotide polymorphisms (SNPs) in known pigmentation genes were tested for association to objective spectrophotometric reflectance measurements of the upper-inner arm and the buttock in 188 unrelated Danes. The 188 Danes were typed for 33 SNPs by two multiplex PCRs, two multiplex single base extension reactions and capillary electrophoresis. To further investigate the role of the SNPs, their allelic distribution was compared to those in a population of 36 Somalis. Four MC1R SNP alleles (R151C, R160W, D294H and 29insA) showed significant associations to light skin pigmentation in individuals who were either homozygous or compound heterozygous. This confirms the regulatory role of MC1R in skin pigmentation. SNP loci in SLC45A2, SLC24A5 and TYR showed large allele frequency differences between Somalis and Danes, which suggested a possible role in skin pigmentation. The average buttock pigmentation was significantly lighter than the average upper-inner arm pigmentation. Therefore, the buttock reflectance measurements better resemble the constitutive skin colour compared to upper-inner arm reflectance measurements and will be used in future studies. [Copyright &y& Elsevier]

Abstract: Single nucleotide polymorphisms (SNPs) affect the pigmentation of the eyes, skin and hair in humans. Four SNPs located in the pigmentation genes HERC2 (rs12913832), OCA2 (rs1800407), SLC45A2 (rs16891982) and IRF4 (rs12203592) known to be important for eye colour were investigated in a Danish population sample. The rs12913832 homozygote genotype GG alone the highest likelihood ratio (LR)=29.4 for blue versus brown eye colour and, therefore, seems to be a major determinant of eye colour in Danes. Furthermore, it was shown, that the highest LR=37.5 for dark versus light eye colour was found when the genotypes rs12913832 AG and rs1800407 GG were observed. rs16891982 and rs12203592 had no discernable effect on the eye colour in Danes. [Copyright &y& Elsevier]

Abstract: The SNPforID multiplex was tested on typical crime case samples. In a mixture study, both controlled experiments with samples mixed in known ratios and a blind study were conducted. All mixtures were identified, including samples mixed in 1:40 ratios. For the study of degraded DNA, 50 crime case samples were selected. Complete SNP profiles were obtained in 80% of the highly degraded samples and at least 41 SNPs were typed in the last 20%. Furthermore, 30% of the highly degraded samples turned out to be mixtures. [Copyright &y& Elsevier]