Amplification Failure Research Articles

Forensic DNA methylation profiling from minimal traces: How low can we go?

Analysis of human DNA methylation (DNAm) can provide additional investigative leads in crime cases, e.g. the type of tissue or body fluid, the chronological age of an individual, and differentiation between identical twins. In contrast to the genetic profile, the DNAm level is not the same in every cell. At the single cell level, DNAm represents a binary event at a defined CpG site (methylated versus non-methylated). The DNAm level from a DNA extract however represents the average level of methylation of the CpG of interest of all molecules in the forensic sample. The variance of DNAm levels between replicates is often attributed to technological issues, i.e. degradation of DNA due to bisulfite treatment, preferential amplification of DNA, and amplification failure. On the other hand, we show that stochastic variations can lead to gross fluctuation in the analysis of methylation levels in samples with low DNA levels. This stochasticity in DNAm results is relevant since low DNA amounts (1pg – 1ng) is rather the norm than the exception when analyzing forensic DNA samples.This study describes a conceptual analysis of DNAm profiling and its dependence on the amount of input DNA. We took a close look at the variation of DNAm analysis due to DNA input and its consequences for different DNAm-based forensic applications.As can be expected, the 95%-confidence interval of measured DNAm becomes narrower with increasing amounts of DNA. We compared this aspect for two different DNAm-based forensic applications: body fluid identification and chronological age determination. Our study shows that DNA amount should be well considered when using DNAm for forensic applications.

Plasmodium vivax genetic diversity and heterozygosity in blood samples and resulting oocysts at the Thai\u2013Myanmar border

BackgroundPolyclonal blood-stage infections of Plasmodium vivax are frequent even in low transmission settings, allowing meiotic recombination between heterologous parasites. Empirical data on meiotic products are however lacking. This study examined microsatellites in oocysts derived by membrane feeding of mosquitoes from blood-stage P. vivax infections at the Thai–Myanmar border.MethodsBlood samples from patients presenting with vivax malaria were fed to Anopheles cracens by membrane feeding and individual oocysts from midguts were obtained by dissection after 7 days. DNA was extracted from oocysts and parental blood samples and tested by microsatellite analysis.ResultsA focused study of eight microsatellite markers was undertaken for nine blood stage infections from 2013, for which derived oocysts were studied in six cases. One or more alleles were successfully amplified for 131 oocysts, revealing high levels of allelic diversity in both blood and oocyst stages. Based on standard criteria for defining minor alleles, there was evidence of clear deviation from random mating (inbreeding) with relatively few heterozygous oocysts compared to variance across the entire oocyst population (FIT = 0.89). The main explanation appeared to be natural compartmentalisation at mosquito (FSC = 0.27) and human stages (FCT = 0.68). One single human case produced a total of 431 successfully amplified loci (across 70 oocysts) that were homozygous and identical to parental alleles at all markers, indicating clonal infection and transmission. Heterozygous oocyst alleles were found at 15/176 (8.5%) successfully amplified loci in the other five cases. There was apparently reduced oocyst heterozygosity in individual oocysts compared to diversity within individual mosquitoes (FIS = 0.55), but this may simply reflect the difficulty of detecting minor alleles in oocysts, given the high rate of amplification failure. Inclusion of minor allele peaks (irrespective of height) when matching peaks were found in related blood or oocyst samples, added 11 minor alleles for 9 oocysts, increasing the number of heterozygous loci to 26/176 (14.8%; p = 0.096).ConclusionThere was an apparently low level of heterozygous oocysts but this can be explained by a combination of factors: relatively low complexity of parental infection, natural compartmentalisation in humans and mosquitoes, and the methodological challenge of detecting minor alleles.

New primers for molecular sex identification of waders

Determining the sex of individuals through molecular methods is an increasingly important research tool, particularly for studies of sexually monomorphic species. However, currently available primer sets to molecularly sex birds are known to cause difficulties in sexing Red Knots Calidris canutus and several other waders, due to amplification failure of, and polymorphisms on, the CHD-Z gene. To counter these problems, we developed a new primer set, 2602F/2669R, and show that these primers can be used to reliably sex Red Knots and eight other wader species in four genera. We expect these primers to be useful in a wide range of wader and possibly non-wader species.

The development of a sensitive droplet digital PCR for quantitative detection of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, resulting in important economic losses in pig farming. Prompt detection of PRRS virus (PRRSV) in the field samples is important for effective PRRS control. Droplet digital PCR (ddPCR) is a novel PCR technology, which offers good precision and direct quantification without using calibration curves. In this study, we established a ddPCR system for the sensitive and accurate quantification of PRRSV. Specificity of the assay was determined by the failure of amplification of other relevant viruses. Quantitative linearity, sensitivity and accuracy of ddPCR were compared to those of real time PCR for PRRSV testing. Both methods showed a high degree of linearity (R2=∼1) and quantitative correlation, although ddPCR showed somewhat higher sensitivity than real time PCR. Collectively, our findings indicate that ddPCR might offer improved analytical sensitivity and specificity for PRRSV measurements.

Drug resistance testing through remote genotyping and predicted treatment options in human immunodeficiency virus type 1 infected Tanzanian subjects failing first or second line antiretroviral therapy.

IntroductionAntiretroviral therapy (ART) has been successfully introduced in low-middle income countries. However an increasing rate of ART failure with resistant virus is reported. We therefore described the pattern of drug resistance mutations at antiretroviral treatment (ART) failure in a real-life Tanzanian setting using the remote genotyping procedure and thereafter predicted future treatment options using rule-based algorithm and the EuResist bioinformatics predictive engine. According to national guidelines, the default first-line regimen is tenofovir + lamivudine + efavirenz, but variations including nevirapine, stavudine or emtricitabine can be considered. If failure on first-line ART occurs, a combination of two nucleoside reverse transcriptase inhibitors (NRTIs) and boosted lopinavir or atazanavir is recommended.Materials and methodsPlasma was obtained from subjects with first (n = 174) or second-line (n = 99) treatment failure, as defined by clinical or immunological criteria, as well as from a control group of ART naïve subjects (n = 17) in Dar es Salaam, Tanzania. Amplification of the pol region was performed locally and the amplified DNA fragment was sent to Sweden for sequencing (split genotyping procedure). The therapeutic options after failure were assessed by the genotypic sensitivity score and the EuResist predictive engine. Viral load was quantified in a subset of subjects with second-line failure (n = 52).ResultsThe HIV-1 pol region was successfully amplified from 55/174 (32%) and 28/99 (28%) subjects with first- or second-line failure, respectively, and 14/17 (82%) ART-naïve individuals. HIV-1 pol sequence was obtained in 82 of these 97 cases (84.5%). Undetectable or very low (<2.6 log10 copies/10−3 L) viral load explained 19 out of 25 (76%) amplification failures in subjects at second-line ART failure. At first and second line failure, extensive accumulation of NRTI (88% and 73%, respectively) and NNRTI (93% and 73%, respectively) DRMs but a limited number of PI DRMs (11% at second line failure) was observed. First line failure subjects displayed a high degree of cross-resistance to second-generation NNRTIs etravirine (ETR; 51% intermediate and 9% resistant) and rilpivirine (RPV; 12% intermediate and 58% resistant), and to abacavir (ABC; 49% resistant) which is reserved for second line therapy in Tanzania. The predicted probability of success with the best salvage regimen at second-line failure decreased from 93.9% to 78.7% when restricting access to the NRTIs, NNRTIs and PIs currently available in Tanzania compared to when including all approved drugs.DiscussionThe split genotyping procedure is potential tool to analyse drug resistance in Tanzania but the sensitivity should be evaluated further. The lack of viral load monitoring likely results in a high false positive rate of treatment failures, unnecessary therapy switches and massive accumulation of NRTI and NNRTI mutations. The introduction of regular virological monitoring should be prioritized and integrated with drug resistance studies in resource limited settings.

Temporal Transcriptomic and Proteomic Landscapes of Deteriorating Pancreatic Islets in Type 2 Diabetic Rats.

Progressive reduction in β-cell mass and function comprise the core of the pathogenesis mechanism of type 2 diabetes. The process of deteriorating pancreatic islets, in which a complex network of molecular events is involved, is not yet fully characterized. We used RNA sequencing and tandem mass tag-based quantitative proteomics technology to measure the temporal mRNA and protein expression changes of pancreatic islets in Goto-Kakizaki (GK) rats from 4 to 24 weeks of age. Our omics data set outlines the dynamics of the molecular network during the deterioration of GK islets as two stages: The early stage (4-6 weeks) is characterized by anaerobic glycolysis, inflammation priming, and compensation for insulin synthesis, and the late stage (8-24 weeks) is characterized by inflammation amplification and compensation failure. Further time course analysis allowed us to reveal 5,551 differentially expressed genes, a large portion of which have not been reported before. Our comprehensive and temporal transcriptome and proteome data offer a valuable resource for the diabetes research community and for quantitative biology.

Comparison of different genetic distances to test isolation by distance between populations.

Studying isolation by distance can provide useful demographic information. To analyze isolation by distance from molecular data, one can use some kind of genetic distance or coalescent simulations. Molecular markers can often display technical caveats, such as PCR-based amplification failures (null alleles, allelic dropouts). These problems can alter population parameter inferences that can be extracted from molecular data. In this simulation study, we analyze the behavior of different genetic distances in Island (null hypothesis) and stepping stone models displaying varying neighborhood sizes. Impact of null alleles of increasing frequency is also studied. In stepping stone models without null alleles, the best statistic to detect isolation by distance in most situations is the chord distance DCSE. Nevertheless, for markers with genetic diversities HS<0.4-0.5, all statistics tend to display the same statistical power. Marginal sub-populations behave as smaller neighborhoods. Metapopulations composed of small sub-population numbers thus display smaller neighborhood sizes. When null alleles are introduced, the power of detection of isolation by distance is significantly reduced and DCSE remains the most powerful genetic distance. We also show that the proportion of null allelic states interact with the slope of the regression of FST/(1-FST) as a function of geographic distance. This can have important consequences on inferences that can be made from such data. Nevertheless, Chapuis and Estoup's FreeNA correction for null alleles provides very good results in most situations. We finally use our conclusions for reanalyzing and reinterpreting some published data sets.

Improved amplification results following episodes of failure to amplify at the Amelogenin Locus using PowerPlex® ESI 16 Fast System

In 2012 the Israel Police DNA Casework laboratory adopted the 16 STR PowerPlex® ESI kit for routine use. The Promega Company updated this kit and developed the PowerPlex® ESI 16 Fast System in which all autosomal primer pairs remained identical to the original set, except at the amelogenin site. The master mix was improved and optimized which allowed for direct, faster and more robust amplification. Prior to implementing the PowerPlex® ESI 16 Fast System in our lab, we conducted a preliminary assay where 213 casework samples were amplified using the new kit. These samples had previously been extracted by one of two extraction kits employed by our lab. (the PrepFiler ExpressTM and PrepFiler BTATM Forensic DNA Extraction Kits). The amplification results from these samples were compared to samples amplified using the original PowerPlex® ESI 16 kit. Multiple incidents of failure to amplify at the amelogenin locus were noted using the new system with the recommended protocol at a rate of 13% (28 of 213 samples). Experiments were performed to understand whether these amplification failures could be a result of primer binding site mutations, extraction method reagents and/or inhibitors. The conclusions reached following these experiments, in conjunction with consultation with the manufacturer, led to the trial of a modified amplification protocol where the suggested annealing temperature was reduced by 2 degrees. To evaluate the efficiency of this altered protocol, a comparison study was undertaken where 88 additional casework samples were chosen and amplified using both the modified 58°C and the recommended 60°C annealing temperatures. We concluded that the most effective method in our laboratory for achieving a consistent and balanced amplification at the amelogenin locus was to reduce the annealing temperature from the manufacturer's recommended 60°C to 58°C. This modification resulted in a reduction of the failure to amplify at the amelogenin locus from 13% (28/213) to 1.1% (1/88) without any observed changes to the autosomal STR amplification results.

Validating the use of non-invasively sourced DNA for population genetic studies using pedigree data

Abstract. Non-invasive genetic sampling has provided valuable ecological data for many species – data which may have been unobtainable using invasive sampling methods. However, DNA obtained non-invasively may be prone to increased levels of amplification failure and genotyping error. Utilizing genotype data from 32 pedigreed koalas, this study aimed to validate the reliability of final consensus genotypes obtained using DNA isolated from koala scats. Pedigree analysis, duplicate genotyping, analysis of mismatched loci and tests for null alleles were used to look for evidence of errors. All genetically confirmed parent–offspring relationships were found to follow Mendelian rules of inheritance. Duplicate genotypes matched in all cases and there was no evidence of null alleles. Related individuals always had different 12-marker genotypes having a minimum of three unique loci (in one full sibling pair), a mode of seven unique loci and a maximum of 11 unique loci. This study demonstrates the capacity of DNA recovered from koala scats to provide reliable genotypes that can unequivocally discriminate individuals and infer parentage, provided data are missing from no more than two loci. Validating data obtained using non-invasive sampling is an important step, allowing potential problems to be identified at an early stage.

Depletion of tumor suppressor Kank1 induces centrosomal amplification via hyperactivation of RhoA

Chromosome instability, frequently found in cancer cells, is caused by a deficiency in cell division, including centrosomal amplification and cytokinesis failure, and can result in abnormal chromosome content or aneuploidy. The small GTPase pathways have been implicated as important processes in cell division. We found that knockdown of a tumor suppressor protein Kank1 increases the number of cells with a micronucleus or bi-/multi-nuclei, which was likely caused by centrosomal amplification. Kank1 interacts with Daam1, known to bind to and activate a small GTPase, RhoA, in actin assembly. Knockdown of Kank1 or overexpression of Daam1, respectively, hyperactivates RhoA, potentially leading to the modulation of the activity of Aurora-A, a key regulator of centrosomal functions, eventually resulting in centrosomal amplification. Kank1 is also associated with contractile ring formation in collaboration with RhoA, and its deficiency results in the interruption of normal daughter cell separation, generating multinucleate cells. Such abnormal segregation of chromosomes may cause further chromosomal instability and abnormal gene functions, leading to tumorigenesis. Thus, Kank1 plays a crucial role in regulating the activity of RhoA through retrieving excess Daam1 and balancing the activities of RhoA and its effectors.

Optimizing direct amplification of forensic commercial kits for STR determination

Direct DNA amplification in forensic genotyping reduces analytical time when large sample sets are being analyzed. The amplification success depends mainly upon two factors: on one hand, the PCR chemistry and, on the other, the type of solid substrate where the samples are deposited. We developed a workflow strategy aiming to optimize times and cost when starting from blood samples spotted onto diverse absorbent substrates. A set of 770 blood samples spotted onto Blood cards, Whatman® 3 MM paper, FTA™ Classic cards, and Whatman® Grade 1 was analyzed by a unified working strategy including a low-cost pre-treatment, a PCR amplification volume scale-down, and the use of the 3500 Genetic Analyzer as the analytical platform. Samples were analyzed using three different commercial multiplex STR direct amplification kits. The efficiency of the strategy was evidenced by a higher percentage of high-quality profiles obtained (over 94%), a reduced number of re-injections (average 3.2%), and a reduced amplification failure rate (lower than 5%). Average peak height ratio among different commercial kits was 0.91, and the intra-locus balance showed values ranging from 0.92 to 0.94. A comparison with previously reported results was performed demonstrating the efficiency of the proposed modifications. The protocol described herein showed high performance, producing optimal quality profiles, and being both time and cost effective.

Effects of time and environmental conditions on the quality of DNA extracted from fecal samples for genotyping of wild deer in a warm temperate broad-leaved forest

Extraction of DNA from non-invasive samples (feces) has been used increasingly in genetic research on wildlife. For effective and reliable genetic analyses, knowledge about which samples should be selected in the field is essential. For this reason, we examined the process of DNA degradation in feces of deer. We collected fresh fecal pellets from three wild deer living in a warm temperate forest. We then assessed the effects of time (3, 5, and 10 days) under three environmental conditions (on the forest floor, on exposed ground, and inside the laboratory) on the rates of correct genotyping (CG), amplification failure (NA), genotyping error among positive amplification (ER), false alleles (FA), and allelic dropout (AD) of 15 microsatellite loci. The rate of CG significantly decreased, and those of NA and FA increased with increasing lapse of time. Rates of CG tended to be highest and those of NA, ER, FA, and AD to be lowest in feces kept inside, followed by those on the forest floor. Suitability of samples for DNA extraction was lowest in fecal pellets left on exposed ground, and we suspect that rain may hasten DNA degradation. NA rate could serve as a reliable indicator of the quality of fecal pellets because it was significantly positively correlated with ER rate. For efficient genetic analyses using deer feces in warm temperate zones, we recommend collecting fecal pellets within 3 days of defecation, during periods without rainfall and from under the cover of trees.

Next-generation sequencing of mixed genomic DNA allows efficient assembly of rearranged mitochondrial genomes in Amolops chunganensis and Quasipaa boulengeri.

Recent improvements in next-generation sequencing (NGS) technologies can facilitate the obtainment of mitochondrial genomes. However, it is not clear whether NGS could be effectively used to reconstruct the mitogenome with high gene rearrangement. These high rearrangements would cause amplification failure, and/or assembly and alignment errors. Here, we choose two frogs with rearranged gene order, Amolops chunganensis and Quasipaa boulengeri, to test whether gene rearrangements affect the mitogenome assembly and alignment by using NGS. The mitogenomes with gene rearrangements are sequenced through Illumina MiSeq genomic sequencing and assembled effectively by Trinity v2.1.0 and SOAPdenovo2. Gene order and contents in the mitogenome of A. chunganensis and Q. boulengeri are typical neobatrachian pattern except for rearrangements at the position of “WANCY” tRNA genes cluster. Further, the mitogenome of Q. boulengeri is characterized with a tandem duplication of trnM. Moreover, we utilize 13 protein-coding genes of A. chunganensis, Q. boulengeri and other neobatrachians to reconstruct the phylogenetic tree for evaluating mitochondrial sequence authenticity of A. chunganensis and Q. boulengeri. In this work, we provide nearly complete mitochondrial genomes of A. chunganensis and Q. boulengeri.

Retrieval of hundreds of nuclear loci from herbarium specimens

AbstractHerbaria are unparalleled collections of biodiversity information representing the world's flora. However, this treasure has remained largely inaccessible to genetic studies, frequently limited by the low yields of poor‐quality DNA. Next‐generation sequencing (NGS) has transformed every field of biological research. The different strategies for accessing genetic data using NGS are changing the direction of biodiversity research—we are no longer constrained by a relatively small number of markers for non‐model organisms, by time and cost limited sample sizes, or by incomplete datasets due to recalcitrant DNA extractions or PCR amplification failure. Here we show that targeted enrichment through hybrid capture can be used to generate hundreds of kilobases of nuclear sequence data of the Neotropical genus Inga, from herbarium specimens as old as 180 years and using as little as 16 ng of degraded DNA.

Detection of Theileria orientalis in mosquito blood meals in the United Kingdom

Theileria spp. are tick-borne protozoan parasites that infect a wide range of wild and domestic animals. In this study, the utility of xenosurveillance of blood-fed specimens of Culiseta annulata for detecting the presence of piroplasms in livestock was investigated. Blood-fed mosquitoes were collected at Elmley National Nature Reserve, Kent, United Kingdom. All specimens were morphologically identified, and DNA barcoding was used to confirm the morphological identification. Both the vertebrate host species and Theileria genome was detected within the bloodmeal by real-time PCR. Sequencing was used to confirm the identity of all amplicons. In total, 105 blood-fed mosquitoes morphologically identified as Cs. annulata were collected. DNA barcoding revealed that 102 specimens were Cs. annulata (99%), while a single specimen was identified as Anopheles messeae. Two specimens could not be identified molecularly due to PCR amplification failure. Blood meal analysis revealed that Cs. annulata fed almost exclusively on cattle at the collection site (n=100). The application of a pan-piroplasm PCR detected 16 positive samples (15.2%) and sequence analysis of the amplicons demonstrated that the piroplasms present in the blood meal belonged to the Theileria orientalis group. This study demonstrates how xenosurveillance can be applied to detecting pathogens in livestock and confirms the presence of Theileria species in livestock from the United Kingdom.

European study on Pneumocystis jirovecii short tandem repeats genotyping reveals wide population diversity with geographic specificities

Introduction Pneumocystis jirovecii is a human specific uncultivable ascomycetous fungus, for which the reservoir is immunocompetent human individuals. Immunocompromised patients are at risk of developing pneumocystis pneumonia (PCP) when exposed to P.jirovecii through their immediate environment. A recently described short tandem repeat (STR) typing strategy was applied to a population of patients from Paris and found a high diversity of genotypes (Gts) between the patients, but identical Gts reflecting putative interhuman nosocomial transmission [1] . In contrast, another genotyping study using a different STR set described a limited global population of P.jirovecii testing isolates recovered from Africa (n = 13), USA (n = 49) and Europe (n = 29) [2] . Our objective is to determine the distribution of P.jirovecii STR genotypes across European hospitals. Methods We investigated a collection of 355 P.jirovecii microscopy or PCR positive respiratory samples recovered from 355 PCP patients in 12 European countries [France (n = 5), Belgium, The Netherlands, Denmark, Germany, UK, Poland, Czech republic, Switzerland, Italy, Spain, and Portugal]. STR typing was performed as previously described [1] . Amplification failure occurred in 32% of the samples, likely a result of insufficient P.jirovecii DNA. Therefore, 242 samples (median: 17 per center (8–24)) were further analyzed. Results Mixtures of STR markers >1 allele for ≥ 1 locus) were detected in 66.7% [range: 36.4%–90.9%] of the samples, with a trend towards a lower proportion of mixtures in France-centre 2 and Belgium. The distribution of alleles in all six markers was significantly different according to the countries in STRPj_138 (p = 0.0002), STRPj_278 (p = 0.0085), and STRPj_279 (p = 0.0069). Genotyping was analyzed only in samples harboring one allele/locus (n = 87) or several alleles for one locus only (n = 56). This provided 200 analyzable combinations corresponding to 143 Gts. Of them, 123 were found only in one country, 16 in two, 2 in three and one in 4 and 5 countries. Nine Gts were found more than once in a given country. Gt123 was significantly associated with France (14/15, p = 0.0007) and Gt132 with Belgium (5/5, p Conclusion Our study of 16 European centers showed a wide population diversity across Europe. However, focusing on centers, our results evidenced clusters of patients harboring a given genotype suggesting nosocomial interhuman transmission and potential outbreak situations.

Fur and faeces: an experimental assessment of non-invasive DNA sampling for the European pine marten

Non-invasive genetic sampling using materials such as faeces or hair can be used to monitor wildlife populations, although DNA quality is often poor. Improving sampling efficiency and minimising factors that reduce DNA quality are therefore critical. After a severe decline, the European pine marten, Martes martes, has reclaimed much of its former range in Scotland, UK. Recording this rapid range expansion requires developing techniques for accurate monitoring, but this is hampered by the species’ elusive behaviour. We tested two sampling methods, hair collected from hair tubes and faeces (scat) collected along tracks, to assess the effects of key environmental and sampling variables on DNA quality and sampling efficiency. For hair, we tested the influence of hair tube location (distance from forest tracks) on collection rate and sex ratio of animals successfully sampled. For scats, we assessed the effect of time since defecation (1 to 16 days) on genotyping error rates and success under two contrasting environmental conditions (exposed to rainfall or sheltered). We found no bias in the collection rate or sex ratio of animals detected by hair samples with differing proximity to forest tracks. DNA amplification failure for scats exposed to rainfall increased from 28 to 65 % over the 16-day experimental period. During periods of low rainfall, the length of collection sessions could therefore be extended to increase sample number without risk of DNA degradation. Lack of bias in hair collection rates with proximity to forest tracks provides justification for tube placement close to tracks, as this reduces survey effort. These findings provide guidance for the development of efficient and cost-effective non-invasive sampling of Scottish pine martens.

Alu SINE analyses of 3,000-year-old human skeletal remains: a pilot study.

BackgroundAs Short Interspersed Elements (SINEs), human-specific Alu elements can be used for population genetic studies. Very recent inserts are polymorphic within and between human populations. In a sample of 30 elements originating from three different Alu subfamilies, we investigated whether they are preserved in prehistorical skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. In the present study, we examined a prehistoric triad of father, mother and daughter.ResultsFor 26 of the 30 Alu loci investigated, definite results were obtained. We were able to demonstrate that presence/absence analyses of Alu elements can be conducted on individuals who lived 3,000 years ago. The preservation of the ancient DNA (aDNA) is good enough in two out of three ancient individuals to routinely allow the amplification of 500 bp fragments. The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures. We here present an alternative molecular approach to deal with these degradation phenomena by using internal Alu subfamily specific primers producing short fragments of approximately 150 bp.ConclusionsOur data clearly show the possibility of presence/absence analyses of Alu elements in individuals from the Lichtenstein cave. Thus, we demonstrate that our method is reliably applicable for aDNA samples with good or moderate DNA preservation. This method will be very useful for further investigations with more Alu loci and larger datasets. Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-016-0063-y) contains supplementary material, which is available to authorized users.

Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification

To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR). Prospective and blinded. Not applicable. Couples indicated for preimplantation genetic diagnosis (PGD). None. Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening. The ADO frequency on a single cell from a fibroblast cell line was 1.64% (18/1,096). When two or more cells were tested, the ADO frequency dropped to 0.02% (1/4,426). The rate of amplification failure was 1.38% (55/4,000) overall, with 2.5% (20/800) for single cells and 1.09% (35/3,200) for samples that had two or more cells. Among 152 embryos tested in 17 cases by qPCR-based PGD and CCS, 100% were successfully given a diagnosis, with 0% ADO or amplification failure. Genotyping consistency with reference laboratory results was >99%. Another 304 embryos from 43 cases were included in the clinical application of qPCR-based PGD and CCS, for which 99.7% (303/304) of the embryos were given a definitive diagnosis, with only 0.3% (1/304) having an inconclusive result owing to recombination. In patients receiving a transfer with follow-up, the pregnancy rate was 82% (27/33). This study demonstrates that the use of qPCR for PGD testing delivers consistent and more reliable results than existing methods and that single gene disorder PGD can be run concurrently with CCS without the need for additional embryo biopsy or whole genome amplification.

Population Genetics and Reproductive Strategies of African Trypanosomes: Revisiting Available Published Data.

Trypanosomatidae are a dangerous family of Euglenobionta parasites that threaten the health and economy of millions of people around the world. More precisely describing the population biology and reproductive mode of such pests is not only a matter of pure science, but can also be useful for understanding parasite adaptation, as well as how parasitism, specialization (parasite specificity), and complex life cycles evolve over time. Studying this parasite’s reproductive strategies and population structure can also contribute key information to the understanding of the epidemiology of associated diseases; it can also provide clues for elaborating control programs and predicting the probability of success for control campaigns (such as vaccines and drug therapies), along with emergence or re-emergence risks. Population genetics tools, if appropriately used, can provide precise and useful information in these investigations. In this paper, we revisit recent data collected during population genetics surveys of different Trypanosoma species in sub-Saharan Africa. Reproductive modes and population structure depend not only on the taxon but also on the geographical location and data quality (absence or presence of DNA amplification failures). We conclude on issues regarding future directions of research, in particular vis-à-vis genotyping and sampling strategies, which are still relevant yet, too often, neglected issues.