Half-volume validation of the NGM Detect™ PCR Amplification Kit and its application on degraded casework samples.

The NGM Detect™ PCR Amplification Kit was designed particularly for genotyping degraded casework samples. This study aimed to validate the half-volume amplification of the kit and to present its successful long-term application. The validation was performed in accordance to the corresponding guidelines of the Scientific Working Group on DNA analysis methods and the European Network of Forensic Science Institutes. For validation parameters, such as sensitivity, reproducibility, and repeatability, polymerase chain reactions (PCR) were set up both manually and robotically, applying 29 cycles. For PCRs with sub-optimal DNA input (≤0.5 ng) the cycle numbers were increased to 31. Regardless of the PCR preparation method, the optimal 0.5 ng DNA input produced optimal allelic peak heights with no allelic dropout. The first alleles that failed to amplify started to appear at the level of 0.0375 ng input DNA, although the manually prepared PCRs produced fewer missing alleles. In this case, the raised cycle number produced 1.9% and 4.4% of dropout for manually and for robotically set up PCRs, respectively. In the case of 84 degraded casework samples, PCRs were prepared only by hand. The kit was able to provide informative profiles for 78.57%, 70.37%, and 69.77% for lowly, moderately, and highly degraded samples, respectively. Allelic dropouts were 26.05%, 44.88%, and 51.23% for the same groups. According to our results, we strongly recommend using the NGM Detect™ Kit in half-volume PCR system and encourage the usage of the kit in the particular cases when other kits fail to produce a complete DNA profile.

Forensic Implications of the Discrepancies Caused between NGS and CE Results by New Microvariant Allele at Penta E Microsatellite.

Examination of STR markers using the MPS technology is becoming more common in forensic genetics, but scientists still have insufficient experience in dealing with ambiguous results. However, it is always essential to resolve discordant data if we want to use the technology as an accredited method in routine forensic casework. During the internal laboratory validation of the Precision ID GlobalFiler NGS STR Panel v2 kit, we observed two discrepant genotypes at Penta E locus compared to the previous capillary electrophoresis results. Each NGS software that we applied (i.e., Converge, STRaitRazor and IGV) returned the same 12,14 and 12,16 genotypes in the two samples, respectively, instead of the 11.3,14 and 11.3,16 genotypes previously observed with CE (Capillary electrophoresis) typing. In the case of the length variant 11.3 alleles, traditional Sanger sequencing confirmed a complete twelve repeat unit structure in both samples. However, after sequencing was extended to the flanking regions of the variant alleles, sequence data revealed a two-bases GG deletion downstream of the last TCTTT repeat motif in the forward strand. The determined allele variant has not been previously reported in the scientific literature and highlights the need for a careful evaluation and thorough concordance studies before using NGS STR data in forensic cases.

Contrasting mtDNA and microsatellite data of great reed warbler Acrocephalus arundinaceus breeding populations on a small geographic scale.

The great reed warbler has two genetically distinguishable haplogroups: "Clade A" occurs in higher proportions in Western Europe and Kazakhstan, and colonised Europe and Asia from a refugium in South-West Europe; and "Clade B", which is more common in Eastern Europe, and colonised parts of Europe from a refugium in the Middle East. Our aims were (i) to analyse the rate of differentiation in Hungarian breeding populations in order to see whether European-scale pattern is visible or not on as a small scale as the territory of Hungary and (ii) to compare the results obtained with mtDNA sequencing and microsatellite markers. To analyse the genetic differentiation, the mtDNA control region II was sequenced in 68 adult breeding birds, and 51 were fingerprinted at 11 microsatellite loci, while both analyses were performed on 36 birds (a total of 83 birds). The microsatellite data gave a better resolution and represented the fine-scale pattern of the suspected recolonisation. The lack of genetic differentiation among the breeding populations based on mitochondrial data seems to support this finding, because the admixture of the clades in this particular geographic region obliterates differentiation. Accordingly, the Fst values from different branches are significantly based on microsatellite data only. The mtDNA methods only give reliable results when a geographic and ecological factor plays a role in the population subdivision, but in the case of an intermixing population larger-scale studies are needed.

Phylogeography of a habitat specialist with high dispersal capability: the Savi's Warbler Locustella luscinioides.

In order to describe the influence of Pleistocene glaciations on the genetic structure and demography of a highly mobile, but specialized, passerine, the Savi's Warbler (Locustella luscinioides), mitochondrial DNA sequences (ND2) and microsatellites were analysed in c.330 individuals of 17 breeding and two wintering populations. Phylogenetic, population genetics and coalescent methods were used to describe the genetic structure, determine the timing of the major splits and model the demography of populations. Savi's Warblers split from its sister species c.8 million years ago and have two major haplotype groups that diverged in the early/middle Pleistocene. One of these clades originated in the Balkans and is currently widespread, showing strong evidence for population expansion; whereas the other is restricted to Iberia and remained stable. Microsatellites agreed with a genetic break around the Pyrenees, but showed considerable introgression and a weaker genetic structure. Both genetic markers showed an isolation-by-distance pattern associated with the population expansion of the eastern clade. Breeding populations seem to be segregated at the wintering sites, but results on migratory connectivity are preliminary. Savi's Warbler is the only known migratory bird species in which Iberian birds did not expand beyond the Pyrenees after the last glaciation. Despite the long period of independent evolution of western and eastern populations, complete introgression occurred when these groups met in Iberia. Mitochondrial sequences indicated the existence of refugia-within-refugia in the Iberian Peninsula during the last glacial period, which is surprising given the high dispersal capacity of this species. Plumage differences of eastern subspecies seemed to have evolved recently through natural selection, in agreement with the glacial expansion hypothesis. This study supports the great importance of the Iberian Peninsula and its role for the conservation of genetic variation.

Effects of Consumption of Bt-maize (MON 810) on the Collembolan Folsomia candida, Over Multiple Generations: A Laboratory Study.

The effect of long-term feeding on Bt-maize by collembolans in the laboratory is virtually unestablished. That is why the aim of the present study was to test whether the reproduction, fecal pellet production or food preference of the collembolan F. candida is affected when fed on Bt-maize for several consecutive generations. The collembolans were fed with Bt-maize for 0, 6, 16 and 22 months and the number of eggs and fecal pellets were determined. The experiment was repeated seven months later with the same populations. Food preference tests were additionally performed. Significant differences were found in food consumption, egg production and food preference between populations in some cases, but no time-response effect was observed. In conclusion, several generations feeding of F. candida on Cry1Ab toxin containing Bt-maize seems not to be harmful to this collembolan species.