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Peter Gill - One of the best experts on this subject based on the ideXlab platform.
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further comment on Low Copy Number typing has yet to achieve general acceptance by budowle b et al 2009 forensic sci int genetics supplement series 2 551 552
Forensic Science International-genetics, 2011Co-Authors: John Buckleton, Peter GillAbstract:This paper provides further comment on “Low Copy Number typing has yet to achieve “general acceptance” ” by Budowle, B., et al, 2009. Forensic Sci. Int. Genetics: Supplement Series 2, 551–552 http://dx.doi.org/10.1016/j.fsigss.2009.08.082
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a universal strategy to interpret dna profiles that does not require a definition of Low Copy Number
Forensic Science International-genetics, 2010Co-Authors: Peter Gill, John BuckletonAbstract:Abstract In this paper we critically examine the causes of the underlying confusion that relates to the issue of Low-template (LT) DNA profile interpretation. Firstly, there is much difficulty in attempting to distinguish between LT-DNA vs. conventional DNA because there is no discrete ‘cut-off' point that can be reasonably defined or evaluated. LT-DNA is loosely characterised by drop-out (where alleles may be missing) and drop-in (where additional alleles may be present). We have previously described probabilistic methods that can be used to incorporate these phenomena using likelihood ratio (LR) principles. This is preferred to the random man not excluded (RMNE) method, because we cannot identify a coherent way forward within the restrictions provided by this framework. Most LT-DNA profiles are interpreted using a ‘consensus' profile method, we called this the ‘ biological model ', where only those alleles that are duplicated in consecutive tests are reported. We recognise that there is an increased need for probabilistic models to take precedence over the biological model . These models are required for all kinds of DNA profiles, not just those that are believed to be Low-template. We also recognise that there is a need for education and training if the methods we recommend are to be widely introduced.
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locomation a software tool for the analysis of Low Copy Number dna profiles
Forensic Science International, 2007Co-Authors: Peter Gill, Amanda Kirkham, James M CurranAbstract:Previously, the interpretation of Low Copy Number (LCN) STR profiles has been carried out using the biological or 'consensus' method-essentially, alleles are not reported, unless duplicated in separate PCR analyses [P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forens. Sci. Int. 112 (2000) 17-40]. The method is now widely used throughout Europe. Although a probabilistic theory was simultaneously introduced, its time-consuming complexity meant that it could not be easily applied in practice. The 'consensus' method is not as efficient as the probabilistic approach, as the former wastes information in DNA profiles. However, the theory was subsequently extended to alLow for DNA mixtures and population substructure in a programmed solution by Curran et al. [J.M. Curran, P. Gill, M.R. Bill, Interpretation of repeat measurement DNA evidence alLowing for multiple contributors and population substructure, Forens. Sci. Int. 148 (2005) 47-53]. In this paper, we describe an expert interpretation system (LoComatioN) which removes this computational burden, and enables application of the full probabilistic method. This is the first expert system that can be used to rapidly evaluate numerous alternative explanations in a likelihood ratio approach, greatly facilitating court evaluation of the evidence. This would not be possible with manual calculation. Finally, the Gill et al. and Curran et al. papers both rely on the ability of the user to specify two quantities: the probability of allelic drop-out, and the probability of allelic contamination ("drop-in"). In this paper, we offer some guidelines on how these quantities may be specified.
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use of Low Copy Number dna in forensic inference
International Congress Series, 2003Co-Authors: Alex Lowe, Peter Gill, Caroline Murray, P Richardson, R Wivell, Gillian Tully, J P WhitakerAbstract:Since January 1999, the Forensic Science Service has routinely carried out Low Copy Number (LCN) DNA profiling in casework. To support this initiative, research has been carried out to discover the characteristics and limitations of LCN DNA by studying a series of well-defined evidence types, such as latent fingermarks, and by measuring the propensity of donors to deposit DNA onto objects that they have touched. D 2003 Elsevier Science B.V. All rights reserved.
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a comparison of the characteristics of profiles produced with the ampflstr sgm plus multiplex system for both standard and Low Copy Number lcn str dna analysis
Forensic Science International, 2001Co-Authors: J P Whitaker, E A Cotton, Peter GillAbstract:DNA STR profiles have been generated from 1 ng and Low Copy Number (LCN) templates using 28 and 34 cycles of amplification, respectively. Characteristics which facilitate the interpretation of profiles, such as heterozygous balance, allelic dropout and stutter proportions have been quantified. We demonstrate that a reduction in DNA template coupled with an increase in amplification cycle Number produces an increased rate of allelic dropout out which can be correlated to the peak areas of those alleles observed. In addition, the LCN conditions increase the degree of peak area asymmetry observed from heterozygotes and the size range of stutters. Analysis of the data alLows us to develop sets of guidelines appropriate for interpreting both single and mixed DNA profiles.
Bruce Budowle - One of the best experts on this subject based on the ideXlab platform.
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utility of amplification enhancers in Low Copy Number dna analysis
International Journal of Legal Medicine, 2015Co-Authors: Pamela L Marshall, Bruce Budowle, Jonathan L KingAbstract:One parameter that impacts the robustness and reliability of forensic DNA analyses is the amount of template DNA used in the polymerase chain reaction (PCR). With short tandem repeat (STR) typing, Low Copy Number (LCN) DNA samples can present exaggerated stochastic effects during the PCR that result in heterozygote peak height imbalance, allele drop out, and increased stutter. Despite these effects, there has been little progress toward decreasing the formation of stutter products and heterozygote peak imbalance effects during PCR. In an attempt to develop a more robust system that is less refractory to stochastic effects, the PCR additives, betaine, DMSO, PEG, and PCRboost®, were investigated on Low-quantity DNA samples. The effects of the additives were assessed by evaluating STR typing results. Of the four additives, the only positive effects were observed with betaine treatment. Betaine, at a final concentration of 1.25 mol/L, was found to improve the robustness of the amplification, specifically by decreasing stutter in a dual locus system. In contrast, the addition of 1.25 mol/L betaine to commercial STR amplification kits did not affect stutter ratios. However, the addition of betaine did lead to increased yield of PCR products in all commercial kits tested. The results support that betaine can improve amplification efficiency of LCN DNA samples.
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reduction of stutter ratios in short tandem repeat loci typing of Low Copy Number dna samples
Forensic Science International-genetics, 2014Co-Authors: Seung Bum Seo, Bruce Budowle, Jonathan L KingAbstract:A B S T R A C T Increased height of stutter peaks is a phenomenon with Low Copy Number (LCN) short tandem repeat (STR) typing that can impact interpretation. An alternative strategy of Lowering the annealing/extension temperature (LT) at 56 8C was designed to attempt to decrease the heights of stutter peaks. STR typing results were generated in terms of stutter ratios using LT-PCR conditions and compared with data obtained using standard (STD) PCR conditions. DNA samples ranging from 100 to 25 pg were amplified using reagents contained in the AmpF‘STR 1 Identifiler 1 PCR Amplification or AmpF‘STR 1 Identifiler 1 Plus PCR Amplification kits with 32 or 34 PCR cycles. Stutter ratios decreased by an average of 14.7%, 14.9% and 18.1% at 100, 50 and 25 pg of template DNA under LT conditions compared with those of STD conditions in the Identifiler 1 Kit amplified samples. The LT conditions also decreased average stutter ratios by 13.3% compared with those of STD conditions in the Identifiler 1 Plus Kit amplified samples. The overall PCR efficiency obtained with STD and LT conditions with the two STR kits was comparable in terms of the Number of detected alleles, peak heights and peak height ratios. These results support the hypothesis that a Lower temperature annealing/extension step reduces the likelihood of slippage during PCR by enhancing the stability of the DNA polymerase/template DNA complex or the stability of the generated duplex than the conditions of the standard extension step. This stability in turn would result in Lower stutter ratios.
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Low Copy Number typing has yet to achieve general acceptance
Forensic Science International: Genetics Supplement Series, 2009Co-Authors: Bruce Budowle, Arthur J. Eisenberg, Angela Van DaalAbstract:When processing a small Number of starting DNA templates during the PCR, exaggerated stochastic sampling effects will occur, and because of increased sensitivity of detection there is a concomitant increased risk of observing contamination. Caution should be taken with using LCN typing and there should be awareness of the pitfalls that have been encountered by some users of LCN typing. The methodology does not yield reliable results; therefore interpretation strategies have been developed in an attempt to overcome the non-reproducibility of LCN typing. It is troubling that the LCN protocols that are available are scant and leave most of the interpretation to the discretion of the analyst. There is substantial evidence that the interpretation by practitioners often is not based on the results of validation studies and is steeped in practices of bias. In addition, no generally accepted approach(es) to statistical analyses and the values to account for the uncertainty associated with the stochastic phenomena, such as allele drop out, are described.
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Validity of Low Copy Number typing and applications to forensic science
Croatian medical journal, 2009Co-Authors: Bruce Budowle, Arthur J. Eisenberg, Angela Van DaalAbstract:Low Copy Number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are beLow the stochastic threshold for reliable interpretation. There are a Number of methodologies to increase sensitivity of detection to enable LCN typing. These approaches encompass modifications during the polymerase chain reaction (PCR) and/or post-PCR manipulations. Regardless of the manipulations, when processing a small Number of starting templates during the PCR exaggerated stochastic sampling effects will occur. The result is that several phenomena can occur: a substantial imbalance of 2 alleles at a given heterozygous locus, allelic dropout, or increased stutter. With increased sensitivity of detection there is a concomitant increased risk of contamination. Recently, a commission reviewed LCN typing and found it to be "robust" and "fit for purpose." Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing. The findings of the commission seem inconsistent with the nature of LCN typing. While LCN typing is appropriate for identification of missing persons and human remains and for developing investigative leads, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. A more in-depth evaluation by the greater scientific community is warranted. The issues to consider include: training and education, evidence handling and collection procedures, the application or purpose for which the LCN result will be used, the reliability of current LCN methods, replicate analyses, interpretation and uncertainty, report writing, validation requirements, and alternate methodologies for better performance.
Mario Falchi - One of the best experts on this subject based on the ideXlab platform.
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metabolomic profile of Low Copy Number carriers at the salivary α amylase gene suggests a metabolic shift toward lipid based energy production
Diabetes, 2016Co-Authors: Abdelilah Arredouani, Matteo Stocchero, Nicola Culeddu, Julia Elsayed Moustafa, Jea Tiche, Everley Alkau, Thierry Ousseau, Marco Manca, Mario FalchiAbstract:Low serum salivary amylase levels have been associated with a range of metabolic abnormalities, including obesity and insulin resistance. We recently suggested that a Low Copy Number at the AMY1 gene, associated with Lower enzyme levels, also increases susceptibility to obesity. To advance our understanding of the effect of AMY1 Copy Number variation on metabolism, we compared the metabolomic signatures of high- and Low-Copy Number carriers. We analyzed, using mass spectrometry and nuclear magnetic resonance (NMR), the sera of healthy normal-weight women carrying either Low-AMY1 copies (LAs: four or fewer copies; n = 50) or high-AMY1 copies (HAs: eight or more copies; n = 50). Best-fitting multivariate models (empirical P < 1 × 10-3) of mass spectrometry and NMR data were concordant in showing differences in lipid metabolism between the two groups. In particular, LA carriers showed Lower levels of long- and medium-chain fatty acids, and higher levels of dicarboxylic fatty acids and 2-hydroxybutyrate (a known marker of glucose malabsorption). Taken together, these observations suggest increased metabolic reliance on fatty acids in LA carriers through β- and ω-oxidation and reduced cellular glucose uptake with consequent diversion of acetyl-CoA into ketogenesis. Our observations are in line with previously reported delayed glucose uptake in LA carriers after starch consumption. Further functional studies are needed to extrapolate from our findings to implications for biochemical pathways.
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Low Copy Number of the salivary amylase gene predisposes to obesity
Nature Genetics, 2014Co-Authors: Mario Falchi, Julia Elsayed S Moustafa, Petros Takousis, Francesco Pesce, Amelie Bonnefond, Johanna C Anderssonassarsson, Peter H Sudmant, Rajkumar Dorajoo, Mashael Alshafai, Leonardo BottoloAbstract:Common multi-allelic Copy Number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts. Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene (AMY1) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 Copy Number was positively associated with both amylase gene expression (P = 2.31 × 10(-14)) and serum enzyme levels (P 9) and bottom (Copy Number < 4) 10% of the Copy Number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies.
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Low Copy Number of the salivary amylase gene predisposes to obesity
Nature Genetics, 2014Co-Authors: Mario Falchi, Julia Elsayed S Moustafa, Petros Takousis, Francesco Pesce, Amelie Bonnefond, Johanna C Anderssonassarsson, Peter H Sudmant, Rajkumar Dorajoo, Mashael Alshafai, Leonardo BottoloAbstract:Mario Falchi, Philippe Froguel and colleagues report association of a multi-allelic Copy Number variant encompassing the salivary amylase gene AMY1 with body mass index and risk of obesity.
Leonardo Bottolo - One of the best experts on this subject based on the ideXlab platform.
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Low Copy Number of the salivary amylase gene predisposes to obesity
Nature Genetics, 2014Co-Authors: Mario Falchi, Julia Elsayed S Moustafa, Petros Takousis, Francesco Pesce, Amelie Bonnefond, Johanna C Anderssonassarsson, Peter H Sudmant, Rajkumar Dorajoo, Mashael Alshafai, Leonardo BottoloAbstract:Common multi-allelic Copy Number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts. Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene (AMY1) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 Copy Number was positively associated with both amylase gene expression (P = 2.31 × 10(-14)) and serum enzyme levels (P 9) and bottom (Copy Number < 4) 10% of the Copy Number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies.
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Low Copy Number of the salivary amylase gene predisposes to obesity
Nature Genetics, 2014Co-Authors: Mario Falchi, Julia Elsayed S Moustafa, Petros Takousis, Francesco Pesce, Amelie Bonnefond, Johanna C Anderssonassarsson, Peter H Sudmant, Rajkumar Dorajoo, Mashael Alshafai, Leonardo BottoloAbstract:Mario Falchi, Philippe Froguel and colleagues report association of a multi-allelic Copy Number variant encompassing the salivary amylase gene AMY1 with body mass index and risk of obesity.
John Buckleton - One of the best experts on this subject based on the ideXlab platform.
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further comment on Low Copy Number typing has yet to achieve general acceptance by budowle b et al 2009 forensic sci int genetics supplement series 2 551 552
Forensic Science International-genetics, 2011Co-Authors: John Buckleton, Peter GillAbstract:This paper provides further comment on “Low Copy Number typing has yet to achieve “general acceptance” ” by Budowle, B., et al, 2009. Forensic Sci. Int. Genetics: Supplement Series 2, 551–552 http://dx.doi.org/10.1016/j.fsigss.2009.08.082
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validation and development of interpretation guidelines for Low Copy Number lcn dna profiling in new zealand using the ampflstr sgm plus multiplex
Forensic Science International-genetics, 2010Co-Authors: Sue Petricevic, John Buckleton, Jonathan Whitaker, Sue Vintiner, Jayshree Patel, Pauline Simon, Helen Ferraby, Waseem Hermiz, Amanda RussellAbstract:The characteristics of STR profiles produced from approximately 1 ng starting template using the AMPFlSTR SGM Plus multiplex and 28 PCR cycles, are well documented. However, the analysis of samples perceived as Low in starting template (less than 100 pg), and referred to as Low template DNA (LTDNA), can require a test of higher sensitivity in order to achieve successful results. One way of increasing this sensitivity is to increase the Number of PCR amplification cycles from 28 to 34. This type of analysis has become known as Low Copy Number, or LCN, DNA profiling. Amplification of LTDNA under LCN conditions can result in increased incidents of profile characteristics such as allelic 'drop-in' and allelic 'drop-out'. Adopting a testing regime which includes duplicate analysis, and maintaining a laboratory environment of stringent and monitored cleanliness, enables the scientist to identify and control these phenomena for a reliable interpretation of the DNA profiling results. A recent court ruling has questioned the reliability of LCN analysis and commented on the paucity of publications surrounding the validation of the technique. We present data for the LCN validation undertaken in our laboratory, and describe the guidelines and working practices we have developed for the analysis and interpretation of profiles generated after LCN profiling. This study augments the published record relating to LCN validation and should act as a useful guide for other laboratories who are considering implementing LCN profiling.
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a universal strategy to interpret dna profiles that does not require a definition of Low Copy Number
Forensic Science International-genetics, 2010Co-Authors: Peter Gill, John BuckletonAbstract:Abstract In this paper we critically examine the causes of the underlying confusion that relates to the issue of Low-template (LT) DNA profile interpretation. Firstly, there is much difficulty in attempting to distinguish between LT-DNA vs. conventional DNA because there is no discrete ‘cut-off' point that can be reasonably defined or evaluated. LT-DNA is loosely characterised by drop-out (where alleles may be missing) and drop-in (where additional alleles may be present). We have previously described probabilistic methods that can be used to incorporate these phenomena using likelihood ratio (LR) principles. This is preferred to the random man not excluded (RMNE) method, because we cannot identify a coherent way forward within the restrictions provided by this framework. Most LT-DNA profiles are interpreted using a ‘consensus' profile method, we called this the ‘ biological model ', where only those alleles that are duplicated in consecutive tests are reported. We recognise that there is an increased need for probabilistic models to take precedence over the biological model . These models are required for all kinds of DNA profiles, not just those that are believed to be Low-template. We also recognise that there is a need for education and training if the methods we recommend are to be widely introduced.
