Publications by authors named "Manuel Fondevila"

34 Publications

Fitting of the In Vitro Gas Production Technique to the Study of High Concentrate Diets.

Animals (Basel) 2020 Oct 21;10(10). Epub 2020 Oct 21.

Departamento de Producción Animal y Ciencia de los Alimentos, Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA, M. Servet 177, 50013 Zaragoza, Spain.

In vitro rumen fermentation systems are often adapted to forage feeding conditions, with pH values ranging in a range close to neutrality (between 6.5 and 7.0). Several attempts using different buffers have been made to control incubation pH in order to evaluate microbial fermentation under conditions simulating high concentrate feeding, but results have not been completely successful because of rapid exhaustion of buffering capacity. Recently, a modification of bicarbonate ion concentration in the buffer of incubation solution has been proposed, which, together with using rumen inoculum from donor ruminants given high-concentrate diets, allows for mimicking such conditions in vitro. It is important to consider that the gas volume recorded is in part directly produced from microbial fermentation of substrates, but also indirectly from the buffering capacity of the medium. Thus, the contribution of each (direct and indirect) gas source to the overall production should be estimated. Another major factor affecting fermentation is the rate of passage, but closed batch systems cannot be adapted to its consideration. Therefore, a simple semicontinuous incubation system has been developed, which studies the rate and extent of fermentation by gas production at the time it allows for controlling medium pH and rate of passage by manual replacement of incubation medium by fresh saliva without including rumen inoculum. The application of this system to studies using high concentrate feeding conditions will also be reviewed here.
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http://dx.doi.org/10.3390/ani10101935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590040PMC
October 2020

In Vitro Estimation of the Effect of Grinding on Rumen Fermentation of Fibrous Feeds.

Animals (Basel) 2020 Apr 23;10(4). Epub 2020 Apr 23.

Departamento de Producción Animal y Ciencia de los Alimentos, Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA, M. Servet 177, 50013 Zaragoza, Spain.

The fermentation patterns of six fiber sources, soybean hulls (SH), sugarbeet pulp (BP), palm kernel cake (PK), oat hulls (OH), dehydrated alfalfa meal (DA), and barley straw (BS) were evaluated for this study on the effect of their presentation form (non-processed, NP and ground, GR). Substrates were tested in a conventional in vitro batch system, using rumen fluid obtained from ewes fed 0.5 alfalfa hay and 0.5 barley straw. All substrates rendered a higher gas production in GR form ( < 0.05) except for BS but ranked similarly irrespective of the presentation form. Among the substrates, when incubated NP, the highest volume of gas was recorded with BP from 8 h onwards ( < 0.05), whereas OH and BS resulted in the lowest gas volume ( < 0.05). During the first half of the incubation period, methane production was higher in GR than NP ( < 0.05). Among substrates, despite NP or GR, methane production with BP was the highest ( < 0.05). Similarly, the presentation form did not qualitatively affect fermentation, as no differences were observed in volatile fatty acids proportions. The effect of particle size of fibrous substrates does not have a major impact on the rate and extent of the rumen microbial fermentation.
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http://dx.doi.org/10.3390/ani10040732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222778PMC
April 2020

Fermentation Pattern of Several Carbohydrate Sources Incubated in an In Vitro Semicontinuous System with Inocula from Ruminants Given Either Forage or Concentrate-Based Diets.

Animals (Basel) 2020 Feb 6;10(2). Epub 2020 Feb 6.

Departamento de Producción Animal y Ciencia de los Alimentos, Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA, M. Servet 177, 50013 Zaragoza, Spain.

The fermentation pattern of several carbohydrate sources and their interaction with the nature of microbial inoculum was studied. Barley (B), maize (M), sorghum, (S), sugarbeet pulp (BP), citrus pulp (CP) and wheat bran (WB) were tested in an in vitro semicontinuous system maintaining poorly buffered conditions from 0 to 6 h, and being gradually buffered to 6.5 from 8 to 24 h to simulate the rumen pH pattern. Rumen fluid inoculum was obtained from lambs fed with either concentrate and barley straw (CI) or alfalfa hay (FI). The extent of fermentation was higher with CI than FI throughout the incubation (p < 0.05). Among the substrates, S, BP and M maintained the highest pH ( < 0.05), whereas CP recorded the lowest pH with both inocula. Similarly, CP recorded the highest gas volume throughout the incubation, followed by WB and B, and S recorded the lowest volume (p < 0.05). On average, the total volatile fatty acid (VFA), as well as lactic acid concentration, was higher with CP than in the other substrates (p < 0.05). The microbial structure was more affected by the animal donor of inoculum than by the substrate. The in vitro semicontinuous system allows for the study of the rumen environment acidification and substrate microbial fermentation under intensive feeding conditions.
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http://dx.doi.org/10.3390/ani10020261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070664PMC
February 2020

Microbial fermentation of starch- or fibre-rich feeds added with dry or pre-activated Saccharomyces cerevisiae studied in vitro under conditions simulating high-concentrate feeding for ruminants.

J Sci Food Agric 2020 Mar 27;100(5):2236-2243. Epub 2020 Jan 27.

Departamento de Producción Animal y Ciencia de los Alimentos, Instituto Agroalimentario de Aragón (IA2), Universidad de Zaragoza-CITA, Zaragoza, Spain.

Background: To study if the effect on fermentation of yeasts added in ruminant diets can be improved, the effect of adding dry (DY) or pre-activated (AY) Saccharomyces cerevisiae, compared with unsupplemented rumen fluid (CT), on barley grain or sugar beet pulp was evaluated under in vitro high-concentrate fermentative conditions. Yeasts were pre-activated by culturing aerobically at 30 °C for 24 h.

Results: In Experiment 1, AY showed a higher concentration than DY at 6 h incubation (6.83 versus 5.76 log cfu mL ; P = 0.007), differences disappearing at 12 h. This was supported by higher gas production with AY, especially on sugar beet pulp. In Experiment 2, incubation pH was 6.24 and 6.31 respectively for barley and sugar beet pulp at 8 h (P < 0.05), but no effect was recorded at 24 h (6.00 and 5.96; P > 0.05). With sugar beet pulp, gas production promoted by AY was the highest (P < 0.05) in the first 8 h of incubation. However, differences with barley were lower and only detected between AY and CT at 12 h (P < 0.05). Total volatile fatty acids (VFAs) concentration at 8 h followed the same trend, but no differences were detected on molar VFAs profile or lactate concentration. Microbial diversity was more affected by the incubation series than by experimental treatments, and inocula including yeasts (AY, DY) did not differ from unsupplemented rumen liquid.

Conclusions: When pre-activated, the concentration of S. cerevisiae was initially higher and resulted in higher gas volumes, and more on a fibrous (sugar beet pulp) than a starchy (barley) substrate. The response is apparently quantitative, since no major changes were detected on biodiversity or fermentation profile. © 2020 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.10249DOI Listing
March 2020

The influence of feeding behaviour on growth performance, carcass and meat characteristics of growing pigs.

PLoS One 2018 15;13(10):e0205572. Epub 2018 Oct 15.

Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, Legnaro, Padova, Italy.

This study investigated the effect of the feeding behaviour on growth performance, and carcass and meat characteristics of 96 barrows fed ad libitum or restrictively with high or low amino acids (AA) diets according to a 2 × 2 factorial design. The feeding behaviour traits were measured with automated feeders. From 86 kg BW, half of the pigs were given feeds with high indispensable (AA) contents, while the other half received feeds with indispensable AA contents reduced by 9% in early finishing (86-118 kg BW) and by 18% in late finishing (118-145 kg BW). Body lipid and protein retentions were estimated from BW and backfat depth measures recorded at the beginning and end of each period. Pigs were slaughtered at 145 kg BW and carcass and meat quality data were recorded. Phenotypic correlations among feeding behaviours, growth performances, and carcass and meat traits were computed from all the data after adjustment for the effects of feeding treatments. As feeding rate was the behavioural trait most highly correlated with performance and carcass traits, the records of each pig were classified into feeding rate tertiles. Then, the data were statistically analysed using a mixed model, which included feed restriction (FR), AA reduction (AAR), the FR × AAR interaction and the feeding rate tertile as fixed factors, and pen as a random factor. Pigs eating faster (52.1 to 118.9 g/min) had significantly greater final body weights (16%), average daily weight gains (27%), estimated protein gains (22%), estimated lipid retention (46%), carcass weights (16%), weights of lean cuts (14%), weights of fat cuts (21%), proportions of fat in the carcass (14%), and 4% lower proportions of carcass lean cuts than pigs eating slowly (12.6 to 38.2 g/min). Manipulating the eating rate, through management or genetic strategies, could affect feed intake and subsequent growth performance, hence carcass quality, but have little influence on feed efficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205572PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188860PMC
April 2019

Ciliate protozoa of the forestomach of llamas (Lama glama) from locations
at different altitude in Argentina.

Zootaxa 2016 Jan 20;4067(1):49-56. Epub 2016 Jan 20.

Instituto Nacional de Tecnología Agropecuaria INTA, Hurlingham, Argentina; Email: unknown.

This study describes the diversity and concentration of the protozoal population from the forestomach of llamas in Argentina at three altitudinal locations. Protozoal diversity was studied in samples from eight llamas from Hurlingham (Buenos Aires, 43 m altitude), four from Tilcara (Jujuy, 2465 m altitude) and six llamas from Cieneguillas (Jujuy, 3800 m altitude). The total concentrations of protozoa in the forestomach contents were 7.9, 9.1 and 4.1 cells x 104 ml-1 in Hurlingham, Tilcara and Cieneguillas, respectively (P>0.05). Entodinium spp. represented 97.9, 92.3 and 71.4% of the protozoal community in Hurlingham, Tilcara and Cieneguillas, respectively, and the remaining protozoa belonged to the Eudiplodinium genus. Entodinium spp. were identified as E. caudatum (mostly morphotype dubardi), E. longinucleatum, E. parvum, E. bovis, E. exiguum, E. dubardi, and a minor presence of E. bimastus (in three animals) and E. ovibos (in one animal). In regards to the rest of protozoal species, Eudiplodinium maggii is the first reported host record for the genus in llamas. This species was present in the forestomach of 14 out of 18 llamas tested, and in one case it was the unique protozoal species. The vestibuliferids, Dasytricha and Isotricha were absent from the forestomach of llamas. Similarly, other species such as those from the Caloscolex genus, Diplodinium cameli and Entodinium ovumrajae, commonly found in Old World Camelids, were also absent from llamas.
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http://dx.doi.org/10.11646/zootaxa.4067.1.3DOI Listing
January 2016

Inference of Ancestry in Forensic Analysis I: Autosomal Ancestry-Informative Marker Sets.

Methods Mol Biol 2016 ;1420:233-53

Forensic Genetics Unit, Luis Concheiro Institute of Forensic Sciences, Genomic Medicine Group, University of Santiago de Compostela, Galicia, 15782, Spain.

An expanding choice of ancestry-informative marker single nucleotide polymorphisms (AIM-SNPs) is becoming available for the forensic user in the form of sensitive SNaPshot-based tests or in alternative single-base extension genotyping systems (e.g., Sequenom iPLEX) that can be adapted for analysis with SNaPshot. In addition, alternative ancestry-informative variation: Indels and STRs can be analyzed using direct PCR-to-CE techniques that offer the possibility to detect mixed profiles. We review the current forensically viable AIM panels, their optimized PCR multiplexes, and the population differentiation power they offer. We also describe how improved population divergence balance can be achieved with the enlarged multiplex scales of next-generation sequencing approaches to enable analysis of admixed individuals without biased estimation of co-ancestry proportions.
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http://dx.doi.org/10.1007/978-1-4939-3597-0_18DOI Listing
January 2018

SNP Markers as Additional Information to Resolve Complex Kinship Cases.

Transfus Med Hemother 2015 Nov 4;42(6):385-8. Epub 2015 Nov 4.

Molecular Oncology, Portuguese Institute of Oncology, Porto, Portugal; ICBAS, Abel Salazar Institute for the Biomedical Sciences, University of Porto, Porto, Portugal;  LPCC, Research Department-Portuguese League Against Cancer (NRNorte), Porto, Portugal.

Background: DNA profiling with sets of highly polymorphic autosomal short tandem repeat (STR) markers has been applied in various aspects of human identification in forensic casework for nearly 20 years. However, in some cases of complex kinship investigation, the information provided by the conventionally used STR markers is not enough, often resulting in low likelihood ratio (LR) calculations. In these cases, it becomes necessary to increment the number of loci under analysis to reach adequate LRs. Recently, it has been proposed that single nucleotide polymorphisms (SNPs) could be used as a supportive tool to STR typing, eventually even replacing the methods/markers now employed.

Methods: In this work, we describe the results obtained in 7 revised complex paternity cases when applying a battery of STRs, as well as 52 human identification SNPs (SNPforID 52plex identification panel) using a SNaPshot methodology followed by capillary electrophoresis.

Results: Our results show that the analysis of SNPs, as complement to STR typing in forensic casework applications, would at least increase by a factor of 4 total PI values and correspondent Essen-Möller's W value.

Conclusions: We demonstrated that SNP genotyping could be a key complement to STR information in challenging casework of disputed paternity, such as close relative individualization or complex pedigrees subject to endogamous relations.
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http://dx.doi.org/10.1159/000440832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698646PMC
November 2015

Pacifiplex: an ancestry-informative SNP panel centred on Australia and the Pacific region.

Forensic Sci Int Genet 2016 Jan 20;20:71-80. Epub 2015 Oct 20.

Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Spain.

The analysis of human population variation is an area of considerable interest in the forensic, medical genetics and anthropological fields. Several forensic single nucleotide polymorphism (SNP) assays provide ancestry-informative genotypes in sensitive tests designed to work with limited DNA samples, including a 34-SNP multiplex differentiating African, European and East Asian ancestries. Although assays capable of differentiating Oceanian ancestry at a global scale have become available, this study describes markers compiled specifically for differentiation of Oceanian populations. A sensitive multiplex assay, termed Pacifiplex, was developed and optimized in a small-scale test applicable to forensic analyses. The Pacifiplex assay comprises 29 ancestry-informative marker SNPs (AIM-SNPs) selected to complement the 34-plex test, that in a combined set distinguish Africans, Europeans, East Asians and Oceanians. Nine Pacific region study populations were genotyped with both SNP assays, then compared to four reference population groups from the HGDP-CEPH human diversity panel. STRUCTURE analyses estimated population cluster membership proportions that aligned with the patterns of variation suggested for each study population's currently inferred demographic histories. Aboriginal Taiwanese and Philippine samples indicated high East Asian ancestry components, Papua New Guinean and Aboriginal Australians samples were predominantly Oceanian, while other populations displayed cluster patterns explained by the distribution of divergence amongst Melanesians, Polynesians and Micronesians. Genotype data from Pacifiplex and 34-plex tests is particularly well suited to analysis of Australian Aboriginal populations and when combined with Y and mitochondrial DNA variation will provide a powerful set of markers for ancestry inference applied to modern Australian demographic profiles. On a broader geographic scale, Pacifiplex adds highly informative data for inferring the ancestry of individuals from Oceanian populations. The sensitivity of Pacifiplex enabled successful genotyping of population samples from 50-year-old serum samples obtained from several Oceanian regions that would otherwise be unlikely to produce useful population data. This indicates tests primarily developed for forensic ancestry analysis also provide an important contribution to studies of populations where useful samples are in limited supply.
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http://dx.doi.org/10.1016/j.fsigen.2015.10.003DOI Listing
January 2016

Exploration of SNP variants affecting hair colour prediction in Europeans.

Int J Legal Med 2015 Sep 11;129(5):963-75. Epub 2015 Jul 11.

Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, A Coruña, Spain.

DNA profiling is a key tool for forensic analysis; however, current methods identify a suspect either by direct comparison or from DNA database searches. In cases with unidentified suspects, prediction of visible physical traits e.g. pigmentation or hair distribution of the DNA donors can provide important probative information. This study aimed to explore single nucleotide polymorphism (SNP) variants for their effect on hair colour prediction. A discovery panel of 63 SNPs consisting of already established hair colour markers from the HIrisPlex hair colour phenotyping assay as well as additional markers for which associations to human pigmentation traits were previously identified was used to develop multiplex assays based on SNaPshot single-base extension technology. A genotyping study was performed on a range of European populations (n = 605). Hair colour phenotyping was accomplished by matching donor's hair to a graded colour category system of reference shades and photography. Since multiple SNPs in combination contribute in varying degrees to hair colour predictability in Europeans, we aimed to compile a compact marker set that could provide a reliable hair colour inference from the fewest SNPs. The predictive approach developed uses a naïve Bayes classifier to provide hair colour assignment probabilities for the SNP profiles of the key SNPs and was embedded into the Snipper online SNP classifier ( http://mathgene.usc.es/snipper/ ). Results indicate that red, blond, brown and black hair colours are predictable with informative probabilities in a high proportion of cases. Our study resulted in the identification of 12 most strongly associated SNPs to hair pigmentation variation in six genes.
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http://dx.doi.org/10.1007/s00414-015-1226-yDOI Listing
September 2015

Completion of a worldwide reference panel of samples for an ancestry informative Indel assay.

Forensic Sci Int Genet 2015 Jul 25;17:75-80. Epub 2015 Mar 25.

Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Spain.

The use of ancestry informative markers (AIMs) in forensic analysis is of considerable utility since ancestry inference can progress an investigation when no identification has been made of DNA from the crime-scene. Short-amplicon markers, including insertion deletion polymorphisms, are particularly useful in forensic analysis due to their mutational stability, capacity to amplify degraded samples and straightforward amplification technique. In this study we report the completion of H952 HGDP-CEPH panel genotyping with a set of 46 AIM-Indels. The study adds Central South Asian and Middle Eastern population data, allowing a comparison of patterns of variation in Eurasia for these markers, in order to enhance their use in forensic analyses, particularly when combined with sets of ancestry informative SNPs. Ancestry analysis using principal component analysis and Bayesian methods indicates that a proportion of classification error occurs with European-Middle East population comparisons, but the 46 AIM-Indels have the capability to differentiate six major population groups when European-Central South Asian comparisons are made. These findings have relevance for forensic ancestry analyses in countries where South Asians form much of the demographic profile, including the UK, USA and South Africa. A novel third allele detected in MID-548 was characterized - despite a low frequency in the HGDP-CEPH panel samples, it appears confined to Central South Asian populations, increasing the ability to differentiate this population group. The H952 data set was implemented in a new open access SPSmart frequency browser - forInDel: Forensic Indel browser.
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http://dx.doi.org/10.1016/j.fsigen.2015.03.011DOI Listing
July 2015

Development of a forensic skin colour predictive test.

Forensic Sci Int Genet 2014 Nov 10;13:34-44. Epub 2014 Jul 10.

Forensic Genetics Unit, Institute of Forensic Science "Luis Concheiro", University of Santiago de Compostela, Spain.

There is growing interest in skin colour prediction in the forensic field. However, a lack of consensus approaches for recording skin colour phenotype plus the complicating factors of epistatic effects, environmental influences such as exposure to the sun and unidentified genetic variants, present difficulties for the development of a forensic skin colour predictive test centred on the most strongly associated SNPs. Previous studies have analysed skin colour variation in single unadmixed population groups, including South Asians (Stokowski et al., 2007, Am. J. Hum. Genet, 81: 1119-32) and Europeans (Jacobs et al., 2013, Hum Genet. 132: 147-58). Nevertheless, a major challenge lies in the analysis of skin colour in admixed individuals, where co-ancestry proportions do not necessarily dictate any one person's skin colour. Our study sought to analyse genetic differences between African, European and admixed African-European subjects where direct spectrometric measurements and photographs of skin colour were made in parallel. We identified strong associations to skin colour variation in the subjects studied from a pigmentation SNP discovery panel of 59 markers and developed a forensic online classifier based on naïve Bayes analysis of the SNP profiles made. A skin colour predictive test is described using the ten most strongly associated SNPs in 8 genes linked to skin pigmentation variation.
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http://dx.doi.org/10.1016/j.fsigen.2014.06.017DOI Listing
November 2014

"New turns from old STaRs": enhancing the capabilities of forensic short tandem repeat analysis.

Electrophoresis 2014 Nov 16;35(21-22):3173-87. Epub 2014 Jul 16.

Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain.

The field of research and development of forensic STR genotyping remains active, innovative, and focused on continuous improvements. A series of recent developments including the introduction of a sixth dye have brought expanded STR multiplex sizes while maintaining sensitivity to typical forensic DNA. New supplementary kits complimenting the core STRs have also helped improve analysis of challenging identification cases such as distant pairwise relationships in deficient pedigrees. This article gives an overview of several recent key developments in forensic STR analysis: availability of expanded core STR kits and supplementary STRs, short-amplicon mini-STRs offering practical options for highly degraded DNA, Y-STR enhancements made from the identification of rapidly mutating loci, and enhanced analysis of genetic ancestry by analyzing 32-STR profiles with a Bayesian forensic classifier originally developed for SNP population data. As well as providing scope for genotyping larger numbers of STRs optimized for forensic applications, the launch of compact next-generation sequencing systems provides considerable potential for genotyping the sizeable proportion of nucleotide variation existing in forensic STRs, which currently escapes detection with CE.
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http://dx.doi.org/10.1002/elps.201400095DOI Listing
November 2014

Comparison of the genetic background of different Colombian populations using the SNPforID 52plex identification panel.

Int J Legal Med 2014 Jan 12;128(1):19-25. Epub 2013 May 12.

IdentiGEN-Genetic Identification Laboratory and Research Group of Genetic Identification, School of Natural and Exact Sciences (FCEN), University of Antioquia, Calle 67, 53-108, Bloque 7-321, Medellin, Colombia.

Various strategies for analysing SNP markers and genotyping have been published with the goal of obtaining informative profiles from biological samples that contain only small amounts of template and/or degraded DNA. In this study, a multiplex assay of 52 autosomal single-nucleotide polymorphisms (SNPs) was used to analyse 438 individuals from urban populations from different regions of Colombia, as well as a sample of 50 Native American individuals of the Pastos ethnic group from Nariño. To determine if significant differences in these 52 SNPs exist between the distinct regions of Colombia, genetic distance and admixture analyses were performed based on the available data for 17 different Colombian population groups and for population groups from Africa, Europe and America. The results demonstrate significant differences between the populations from the Southwest Andean, Central-West Andean, Central-East Andean, Orinoquian and northern Colombian Pacific Coast regions. Most of the regions exhibited a European and Native American admixture. One exception is the population from the region of Chocó (on the northern Pacific Coast), which exhibits a high proportion of African admixture (54 %). From the observed genetic backgrounds, it is possible to conclude that a single reference database for the entire country would not be suitable for forensic purposes. The allele frequencies and the forensically relevant parameters were calculated for all of the markers in each Colombian region with significant values for the combined matching probability (power of discrimination ≥0.99999999999999990) and the combined probability of exclusion (≥0.9990) in trios that were obtained from all of the population groups.
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http://dx.doi.org/10.1007/s00414-013-0858-zDOI Listing
January 2014

Microbial ecosystem and fermentation traits in the caecum of growing rabbits given diets varying in neutral detergent soluble and insoluble fibre levels.

Anaerobe 2013 Apr 9;20:50-7. Epub 2013 Feb 9.

Instituto Universitario de Investigación en Ciencias Ambientales, Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Miguel Servet 177, 50013, Zaragoza, Spain.

The effect of the level of neutral detergent fibre (NDF: 0.35, LI and 0.42, HI) and neutral detergent soluble fibre (NDSF: 0.14, LS and 0.17, HS) in the caecal ecosystem was studied in 24 weaned (28 days of age) rabbits, weighing 630 ± 80.2 g in a 2 × 2 factorial design. After 22 days, rabbits were slaughtered and their caecal contents sampled. The caecal pH (on average 6.2) and molar volatile fatty acids (VFA) proportions were not affected by dietary treatments, but total VFA concentration tended to be lower with NDF (84.7 vs. 74.1 mmol/l; P = 0.095). The amount of total bacteria tended (P = 0.075) to increase with NDSF, but only in diets with 0.35 NDF. The caecal proportions of Ruminococcus albus and Fibrobacter succinogenes were not affected by type or level of fibre, but Butyrivibrio fibrisolvens decreased (P = 0.055) with the NDF proportion in LS diets. Denaturing gradient gel electrophoresis (DGGE) analysis showed that bacterial communities clustered according to each combination of NDF and NDSF, but did not greatly differ among diets (similarity indexes between 0.67 and 0.70), nor biodiversity was affected (average Shannon and richness indexes 3.50 and 33.1; P > 0.10). Archaeal population revealed changes in the amount and composition that were particularly evident in HS diets, decreasing in concentration (from 4.37 to 4.12 log10 gene copy number/g) and biodiversity (Shannon index from 3.14 to 2.52 and richness index from 23.7 to 13.9) compared to LS. The type and level of dietary fibre had a minor impact on caecal fermentation traits or caecal bacterial community. However, the increase in NDSF from 0.14 to 0.17 reduced concentration and diversity of methanogenic archaea.
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http://dx.doi.org/10.1016/j.anaerobe.2013.02.001DOI Listing
April 2013

Development of a novel forensic STR multiplex for ancestry analysis and extended identity testing.

Electrophoresis 2013 Apr 18;34(8):1151-62. Epub 2013 Mar 18.

Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain.

There is growing interest in developing additional DNA typing techniques to provide better investigative leads in forensic analysis. These include inference of genetic ancestry and prediction of common physical characteristics of DNA donors. To date, forensic ancestry analysis has centered on population-divergent SNPs but these binary loci cannot reliably detect DNA mixtures, common in forensic samples. Furthermore, STR genotypes, forming the principal DNA profiling system, are not routinely combined with forensic SNPs to strengthen frequency data available for ancestry inference. We report development of a 12-STR multiplex composed of ancestry informative marker STRs (AIM-STRs) selected from 434 tetranucleotide repeat loci. We adapted our online Bayesian classifier for AIM-SNPs: Snipper, to handle multiallele STR data using frequency-based training sets. We assessed the ability of the 12-plex AIM-STRs to differentiate CEPH Human Genome Diversity Panel populations, plus their informativeness combined with established forensic STRs and AIM-SNPs. We found combining STRs and SNPs improves the success rate of ancestry assignments while providing a reliable mixture detection system lacking from SNP analysis alone. As the 12 STRs generally show a broad range of alleles in all populations, they provide highly informative supplementary STRs for extended relationship testing and identification of missing persons with incomplete reference pedigrees. Lastly, mixed marker approaches (combining STRs with binary loci) for simple ancestry inference tests beyond forensic analysis bring advantages and we discuss the genotyping options available.
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http://dx.doi.org/10.1002/elps.201200621DOI Listing
April 2013

Characterisation of caecal microbial diversity of lactating does and their offspring given diets with different neutral detergent soluble to insoluble fibre ratios.

Antonie Van Leeuwenhoek 2013 May 26;103(5):1057-68. Epub 2013 Jan 26.

Departamento de Producción Animal y Ciencia de los Alimentos, Instituto Universitario de Investigación en Ciencias Ambientales, Universidad de Zaragoza, Miguel Servet 177, 50013, Zaragoza, Spain.

The effect of neutral detergent soluble fibre (NDSF) to neutral detergent fibre (NDF) dietary ratio (0.29, LR and 0.43, HR) on the caecal ecosystem of lactating does and their offspring was studied. From the 17th day of lactation, each diet was given to four does, allowing for free access to their litters. Does were sampled at 17 and 28 days of lactation, and also two pups per litter at 17 (milk-fed only), 28 (milk and solid fed) and 49 days of age. DGGE was used to study bacterial caecal biodiversity, and total bacterial concentration and relative proportions of Ruminococcus albus and Butyrivibrio fibrisolvens were quantified by real time PCR. In does, diet did not affect (P > 0.10) diversity indexes, total bacterial concentration or relative abundance of B. fibrisolvens, but at 28 days of lactation the proportion of R. albus was higher with LR (interaction Diet × Time, P = 0.037). Caecal communities of pups of 17 days were grouped by litter, but the influence of the mother was reduced at 28 days with solid feed intake, and at 49 days rabbits clustered by diet. Caecal biodiversity increased from 17 to 28 days, and was reduced at 49 days (Shannon index of 3.60, 3.71 and 3.57, respectively; P = 0.049). Total bacterial concentration and relative abundance of R. albus and B. fibrisolvens increased with solid feed intake from 17 to 28 days (P < 0.01), remaining unaffected thereafter. Access of pups to solid feed from 17 days of age modulates the development and composition of the caecal microbiota at weaning.
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http://dx.doi.org/10.1007/s10482-013-9885-5DOI Listing
May 2013

Uniparental markers of contemporary Italian population reveals details on its pre-Roman heritage.

PLoS One 2012 10;7(12):e50794. Epub 2012 Dec 10.

Unidade de Xenética, Facultade de Medicina, Instituto de Medicina Legal, Universidade de Santiago de Compostela, Galicia, Spain.

Background: According to archaeological records and historical documentation, Italy has been a melting point for populations of different geographical and ethnic matrices. Although Italy has been a favorite subject for numerous population genetic studies, genetic patterns have never been analyzed comprehensively, including uniparental and autosomal markers throughout the country.

Methods/principal Findings: A total of 583 individuals were sampled from across the Italian Peninsula, from ten distant (if homogeneous by language) ethnic communities--and from two linguistic isolates (Ladins, Grecani Salentini). All samples were first typed for the mitochondrial DNA (mtDNA) control region and selected coding region SNPs (mtSNPs). This data was pooled for analysis with 3,778 mtDNA control-region profiles collected from the literature. Secondly, a set of Y-chromosome SNPs and STRs were also analyzed in 479 individuals together with a panel of autosomal ancestry informative markers (AIMs) from 441 samples. The resulting genetic record reveals clines of genetic frequencies laid according to the latitude slant along continental Italy--probably generated by demographical events dating back to the Neolithic. The Ladins showed distinctive, if more recent structure. The Neolithic contribution was estimated for the Y-chromosome as 14.5% and for mtDNA as 10.5%. Y-chromosome data showed larger differentiation between North, Center and South than mtDNA. AIMs detected a minor sub-Saharan component; this is however higher than for other European non-Mediterranean populations. The same signal of sub-Saharan heritage was also evident in uniparental markers.

Conclusions/significance: Italy shows patterns of molecular variation mirroring other European countries, although some heterogeneity exists based on different analysis and molecular markers. From North to South, Italy shows clinal patterns that were most likely modulated during Neolithic times.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050794PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3519480PMC
June 2013

Bacterial profile from caecal contents and soft faeces in growing rabbits given diets differing in soluble and insoluble fibre levels.

Anaerobe 2012 Dec 30;18(6):602-7. Epub 2012 Oct 30.

Instituto Universitario de Investigación en Ciencias Ambientales, Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain.

To verify if non-invasive collection of soft faeces (SF) from rabbits can be used as an index of bacterial biodiversity in caecal contents (CC), 24 weaned rabbits were given diets with low (LI) and high (HI) levels of insoluble fibre (neutral detergent fibre, NDF) and low (LS) and high (HS) levels of soluble fibre (neutral detergent soluble fibre, NDSF). After 21 days, animals were fitted with neck collars for SF collection. Two days later, animals were slaughtered and CC sampled. Total bacterial concentration quantified by real time PCR (log(10) ng DNA/mg DM) was higher in SF than CC (2.615 vs. 2.383). Among diets, in CC it was (P = 0.059) lowest in LILS diet, whereas in SF it decreased (P = 0.025) with the NDF level. DGGE profiles showed that structure of bacterial communities of SF was close to that of CC; however, similarity was higher in LI than HI diets (0.82 vs. 0.74). Diversity indexes in CC decreased with NDSF (P < 0.05), whereas the effect of NDF (P < 0.05) was also appreciated in SF. Soft faeces can be an alternative to surgery or slaughter techniques to monitor changes in caecal bacterial community; however, high dietary NDF may decrease similarity between both communities.
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http://dx.doi.org/10.1016/j.anaerobe.2012.10.006DOI Listing
December 2012

Differentiation of African components of ancestry to stratify groups in a case-control study of a Brazilian urban population.

Genet Test Mol Biomarkers 2012 Jun 30;16(6):524-30. Epub 2012 Jan 30.

Faculty of Pharmaceutical Sciences, University of Sao Paulo, São Paulo, Brazil.

Background: Balancing the subject composition of case and control groups to create homogenous ancestries between each group is essential for medical association studies.

Methods: We explored the applicability of single-tube 34-plex ancestry informative markers (AIM) single nucleotide polymorphisms (SNPs) to estimate the African Component of Ancestry (ACA) to design a future case-control association study of a Brazilian urban sample.

Results: One hundred eighty individuals (107 case group; 73 control group) self-described as white, brown-intermediate or black were selected. The proportions of the relative contribution of a variable number of ancestral population components were similar between case and control groups. Moreover, the case and control groups demonstrated similar distributions for ACA <0.25 and >0.50 categories. Notably a high number of outlier values (23 samples) were observed among individuals with ACA <0.25. These individuals presented a high probability of Native American and East Asian ancestral components; however, no individuals originally giving these self-described ancestries were observed in this study.

Conclusions: The strategy proposed for the assessment of ancestry and adjustment of case and control groups for an association study is an important step for the proper construction of the study, particularly when subjects are taken from a complex urban population. This can be achieved using a straight forward multiplexed AIM-SNPs assay of highly discriminatory ancestry markers.
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http://dx.doi.org/10.1089/gtmb.2011.0267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378015PMC
June 2012

Pharmacogenetics of OATP transporters reveals that SLCO1B1 c.388A>G variant is determinant of increased atorvastatin response.

Int J Mol Sci 2011 9;12(9):5815-27. Epub 2011 Sep 9.

Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo 05508-900, Brazil; E-Mails: (P.M.S.P.); (V.N.S.); (F.D.V.G.); (M.A.V.W.); (S.S.A.); (A.D.L.); (M.H.H.); (R.D.C.H.).

Aims: The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated.

Material And Methods: One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot(®) and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (-71T>C) gene polymorphisms were identified by TaqMan(®) Real-time PCR.

Results: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%:1.3-8.0, p < 0.05).

Conclusion: SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy.
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http://dx.doi.org/10.3390/ijms12095815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189752PMC
February 2015

Predation of Salmonella enterica serovar Typhimurium by the rumen protozoon Entodinium caudatum studied in vitro by fluorescence emission.

Eur J Protistol 2010 Aug 27;46(3):189-95. Epub 2010 Apr 27.

Instituto Universitario de Investigación en Ciencias Ambientales, Departamento de Producción Animal y Ciencia de los Alimentos, Miguel Servet 177, 50013 Zaragoza, Spain.

Predation of bacteria by protozoa has important implications on rumen metabolism and bacterial populations. Protozoa can also restrict the passage of pathogenic bacteria to the host's lower gastrointestinal tract. This work aimed to evaluate the predation by Entodinium caudatum (EC) and the intraprotozoal survival of Salmonella enterica serovar Typhimurium. EC cells from a monofaunated sheep were incubated for up to 105 min with a S. enterica strain producing a green fluorescent protein. Rumen fluid from a defaunated sheep (DEF) was used as a control. Fluorescence, as an index of predation, measured in the residual (protozoal) fraction was higher in EC than in DEF. 105 min after the beginning of the incubation it was higher than 30 min after. Intracellular survival of Salmonella within EC was assessed by means of a selective medium. Amounts of Salmonella in the residual fraction were higher in EC than in DEF only after 30 min. After 105 min, each protozoa engulfed 100 Salmonella cell per min. Intraprotozoal survival of ingested Salmonella was 0.0017. Predation of S. enterica by E. caudatum occurred and increased in proportion to time, but bacterial viability inside the protozoa was lower at 105 min. This study demonstrates that fluorescence emission combined with bacterial and protozoal cultures could be a reliable method for quantifying bacterial predation and viability in vitro.
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http://dx.doi.org/10.1016/j.ejop.2010.03.003DOI Listing
August 2010

Ancestry analysis in the 11-M Madrid bomb attack investigation.

PLoS One 2009 Aug 11;4(8):e6583. Epub 2009 Aug 11.

Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Galicia, Spain.

The 11-M Madrid commuter train bombings of 2004 constituted the second biggest terrorist attack to occur in Europe after Lockerbie, while the subsequent investigation became the most complex and wide-ranging forensic case in Spain. Standard short tandem repeat (STR) profiling of 600 exhibits left certain key incriminatory samples unmatched to any of the apprehended suspects. A judicial order to perform analyses of unmatched samples to differentiate European and North African ancestry became a critical part of the investigation and was instigated to help refine the search for further suspects. Although mitochondrial DNA (mtDNA) and Y-chromosome markers routinely demonstrate informative geographic differentiation, the populations compared in this analysis were known to show a proportion of shared mtDNA and Y haplotypes as a result of recent gene-flow across the western Mediterranean, while any two loci can be unrepresentative of the ancestry of an individual as a whole. We based our principal analysis on a validated 34plex autosomal ancestry-informative-marker single nucleotide polymorphism (AIM-SNP) assay to make an assignment of ancestry for DNA from seven unmatched case samples including a handprint from a bag containing undetonated explosives together with personal items recovered from various locations in Madrid associated with the suspects. To assess marker informativeness before genotyping, we predicted the probable classification success for the 34plex assay with standard error estimators for a naïve Bayesian classifier using Moroccan and Spanish training sets (each n = 48). Once misclassification error was found to be sufficiently low, genotyping yielded seven near-complete profiles (33 of 34 AIM-SNPs) that in four cases gave probabilities providing a clear assignment of ancestry. One of the suspects predicted to be North African by AIM-SNP analysis of DNA from a toothbrush was identified late in the investigation as Algerian in origin. The results achieved illustrate the benefit of adding specialized marker sets to provide enhanced scope and power to an already highly effective system of DNA analysis for forensic identification.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006583PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719087PMC
August 2009

Rumen protozoal diversity in the Spanish ibex (Capra pyrenaica hispanica) as compared with domestic goats (Capra hircus).

Eur J Protistol 2009 May 16;45(2):112-20. Epub 2008 Oct 16.

Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Zaragoza, Spain.

Rumen protozoal diversity in the Spanish ibex (SI) was studied in males (n=4), females (n=7) and young (n=4) from the Maestrazgo (Spain) and contrasted with domestic goats (n=3; DG) of the same region. There were no differences among SI types in protozoal concentration or in the number of protozoal species. Only protozoa from the genus Entodinium were observed in SI (seven species), the highest numbers corresponding to E. damae, E. ovibos and E. parvum. DG harboured threefold more species than SI. Nine to 10 Entodinium spp. were observed, but E. ovibos was absent from the rumen of DG, and E. damae was in only one animal. E. caudatum (caudatum, dubardi and lobospinosum morphotypes) occurred in the highest percentage, and E. dubardi, E. exiguum and E. parvum were quite abundant. Four genera of the subfamily Diplodiniinae and the genera Isotricha and Dasytricha from the family Isotrichidae were detected in DG. Epidinium (two DG) and Ophryoscolex (one DG) were also observed. Denaturing gradient gel electrophoresis analysis agreed with microscopic classification, showing up to 8 and 16 bands in SI and DG samples, respectively. The three DG clustered together (similarity index over 0.84), and separately from SI (similarity index over 0.86), with only 0.58 similarity between host species.
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http://dx.doi.org/10.1016/j.ejop.2008.07.001DOI Listing
May 2009

Contribution of gut microbial lysine to liver and milk amino acids in lactating does.

Br J Nutr 2008 Nov 12;100(5):977-83. Epub 2008 Mar 12.

Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, 50013, Spain.

The contribution of microbial amino acids through caecotrophy to tissue protein metabolism was investigated in lactating does. Attempts were made to vary microbial supply through a dietary antibiotic, Zn bacitracin, and to vary tissue demand through manipulation of litter size. Three groups of eight New Zealand does were fed different experimental diets from day 28 of pregnancy to day 26 of lactation. The control group received the basal diet formulated to meet requirements with grass hay, wheat, soybean meal and barley grain. The second (no antibiotic) group and the third (bacitracin; BAC) group ingested the basal diet supplemented with ammonium sulfate (5 g/kg), initially unlabelled (day 1 to day 8) then labelled with 15N (day 9 to day 30), while the BAC diet was also supplemented throughout with antibiotic (Zn bacitracin; 100 mg/kg). From just after birth each group of does was subdivided into two groups, each of four females, with the litter size either five (LS5) or nine (LS9) pups. The 15N enrichment in liver, milk and caecal bacteria amino acids was determined by GC-combustion-isotope ratio MS. All amino acids in bacterial protein were enriched with the (15 NH 4)2SO4 treatment, with lysine 15N enrichment significantly greater in caecal bacteria (0.23 (SE 0.0063) atom % excess (ape)) than in liver (0.04 (SE 0.0004) ape) or milk protein (0.05 (SE 0.0018) ape), confirming the double origin (bacterial and dietary) of tissue lysine. The contribution of microbes to tissue lysine was 0.23 (SE 0.006) when milk protein was used as reference.
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http://dx.doi.org/10.1017/S0007114508957986DOI Listing
November 2008

Forensic validation of the SNPforID 52-plex assay.

Forensic Sci Int Genet 2007 Jun 6;1(2):186-90. Epub 2007 Mar 6.

Centre for Haematology, ICMS, Barts & the London, Queen Mary's School of Medicine & Dentistry, London, United Kingdom.

The advantages of single nucleotide polymorphism (SNP) typing in forensic genetics are well known and include a wider choice of high-throughput typing platforms, lower mutation rates, and improved analysis of degraded samples. However, if SNPs are to become a realistic supplement to current short tandem repeat (STR) typing methods, they must be shown to successfully and reliably analyse the challenging samples commonly encountered in casework situations. The European SNPforID consortium, supported by the EU GROWTH programme, has developed a multiplex of 52 SNPs for forensic analysis, with the amplification of all 52 loci in a single reaction followed by two single base extension (SBE) reactions which are detected with capillary electrophoresis. In order to validate this assay, a variety of DNA extracts were chosen to represent problems such as low copy number and degradation that are commonly seen in forensic casework. A total of 40 extracts were used in the study, each of which was sent to two of the five participating laboratories for typing in duplicate or triplicate. Laboratories were instructed to carry out their analyses as if they were dealing with normal casework samples. Results were reported back to the coordinating laboratory and compared with those obtained from traditional STR typing of the same extracts using Powerplex 16 (Promega). These results indicate that, although the ability to successfully type good quality, low copy number extracts is lower, the 52-plex SNP assay performed better than STR typing on degraded samples, and also on samples that were both degraded and of limited quantity, suggesting that SNP analysis can provide advantages over STR analysis in forensically relevant circumstances. However, there were also additional problems arising from contamination and primer quality issues and these are discussed.
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http://dx.doi.org/10.1016/j.fsigen.2007.01.004DOI Listing
June 2007

Effect of antibiotics on the bacterial population of the rabbit caecum.

FEMS Microbiol Lett 2007 Jul 10;272(2):144-53. Epub 2007 May 10.

Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Spain.

The effect feeding antibiotics has on the bacterial population of the rabbit caecum was investigated. No changes in total volatile fatty acid production or total bacterial counts were observed compared with nonantibiotic treated controls. However, treatment with chlortetracycline resulted in an increase of propionate at the apparent cost of butyrate (P<0.05). Denaturing gradient gel electrophoresis analysis indicated that the two antibiotics that inhibit protein synthesis (chlortetracycline and tiamulin) exerted the most similar changes on the bacterial population structure, decreasing the diversity of the profiles. Sequence analysis of DNA from excised denaturing gradient gel electrophoresis bands was carried out. The majority of the sequences observed were most similar to bacterial sequences previously described in other gut environments, with 11% being most similar to those previously reported from the rabbit, and 95% of the sequences having 95% or greater identity to sequences already in GenBank.
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http://dx.doi.org/10.1111/j.1574-6968.2007.00746.xDOI Listing
July 2007

A multiplex assay with 52 single nucleotide polymorphisms for human identification.

Electrophoresis 2006 May;27(9):1713-24

Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Copenhagen, Denmark.

A total of 52 SNPs reported to be polymorphic in European, Asian and African populations were selected. Of these, 42 were from the distal regions of each autosome (except chromosome 19). Nearly all selected SNPs were located at least 100 kb distant from known genes and commonly used STRs. We established a highly sensitive and reproducible SNP-typing method with amplification of all 52 DNA fragments in one PCR reaction followed by detection of the SNPs with two single base extension reactions analysed using CE. The amplicons ranged from 59 to 115 bp in length. Complete SNP profiles were obtained from 500 pg DNA. The 52 loci were efficiently amplified from degraded samples where previously only partial STR profiles had been obtained. A total of 700 individuals from Denmark, Greenland, Somalia, Turkey, China, Germany, Taiwan, Thailand and Japan were typed, and the allele frequencies estimated. All 52 SNPs were polymorphic in the three major population groups. The mean match probability was at least 5.0 x 10(-19) in the populations studied. Typical paternity indices ranged from 336 000 in Asians to 549 000 in Europeans. Details of the 52 SNP loci and population data generated in this work are freely available at http://www.snpforid.org.
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http://dx.doi.org/10.1002/elps.200500671DOI Listing
May 2006