Second-generation sequencing of forensic STRs using the Ion Torrent™ HID STR 10-plex and the Ion PGM™

Second-generation sequencing (SGS) using Roche/454 and Illumina platforms has proved capable of sequencing the majority of the key forensic genetic STR systems. Given that Roche has announced that the 454 platforms will no longer be supported from 2015, focus should now be shifted to competing SGS platforms, such as the MiSeq (Illumina) and the Ion Personal Genome Machine (Ion PGM™; Thermo Fisher). There are currently several challenges faced with amplicon-based SGS STR typing in forensic genetics, including current lengths of amplicons for CE-typing and lack of uniform data analysis between laboratories.Thermo Fisher has designed a human identification (HID) short tandem repeat (STR) 10-plex panel including amelogenin, CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX and vWA, where the primers have been designed specifically for the purpose of SGS and the data analysis is supported by Ion Torrent™ software. Hence, the combination of the STR 10-plex and the Ion PGM™ represents the first fully integrated SGS STR typing solution from PCR to data analysis.In this study, four experiments were performed to evaluate the alpha-version of the STR 10-plex: (1) typing of control samples; (2) analysis of sensitivity; (3) typing of mixtures; and (4) typing of biological crime case samples. Full profiles and concordant results between replicate SGS runs and CE-typing were observed for all control samples. Full profiles were seen with DNA input down to 50 pg, with the exception of a single locus drop-out in one of the 100 pg dilutions. Mixtures were easily deconvoluted down to 20:1, although alleles from the minor contributor had to be identified manually as some signals were not called by the Ion Torrent™ software. Interestingly, full profiles were obtained for all biological samples from real crime and identification cases, in which only partial profiles were obtained with PCR-CE assays. In conclusion, the Ion Torrent™ HID STR 10-plex panel offers an all-in-one solution from amplification of STRs and amelogenin, and sequencing to data analysis.     

Next generation sequencing of SNPs using the HID-Ion AmpliSeq™ Identity Panel on the Ion Torrent PGM™ platform

The HID-Ion AmpliSeq™ Identity Panel (the HID Identity Panel) is designed to detect 124-plex single nucleotide polymorphisms (SNPs) with next generation sequencing (NGS) technology on the Ion Torrent PGM™ platform, including 90 individual identification SNPs (IISNPs) on autosomal chromosomes and 34 lineage informative SNPs (LISNPs) on Y chromosome. In this study, we evaluated performance for the HID Identity Panel to provide a reference for NGS-SNP application, focusing on locus strand balance, locus coverage balance, heterozygote balance, and background signals. Besides, several experiments were carried out to find out improvements and limitations of this panel, including studies of species specificity, repeatability and concordance, sensitivity, mixtures, case-type samples and degraded samples, population genetics and pedigrees following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. In addition, Southern and Northern Chinese Han were investigated to assess applicability of this panel. Results showed this panel led to cross-reactivity with primates to some extent but rarely with non-primate animals. Repeatable and concordant genotypes could be obtained in triplicate with one exception at rs7520386. Full profiles could be obtained from 100 pg input DNA, but the optimal input DNA would be 1 ng–200 pg with 21 initial PCR cycles. A sample with ≥20% minor contributor could be considered as a mixture by the number of homozygotes, and full profiles belonging to minor contributors could be detected between 9:1 and 1:9 mixtures with known reference profiles. Also, this assay could be used for case-type samples and degraded samples. For autosomal SNPs (A-SNPs), F_ST across all 90 loci was not significantly different between Southern and Northern Chinese Han or between male and female samples. All A-SNP loci were independent in Chinese Han population. Except for 18 loci with H_e <0.4, most of the A-SNPs in the HID Identity Panel presented high polymorphisms. Forensic parameters were calculated as >99.999% for combined discrimination power (CDP), 0.999999724 for combined power of exclusion (CPE), 1.390 × 10¹¹ for combined likelihood ratio (CLR) of trios, and 2.361 × 10⁶ for CLR of motherless duos. For Y-SNPs, a total of 8 haplotypes were observed with the value of 0.684 for haplotype diversity. As a whole, the HID Identity Panel is a well-performed, robust, reliable and high informative NGS-SNP assay and it can fully meet requirements for individual identification and paternity testing in forensic science.     

Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™

Next generation sequencing (NGS) offers the opportunity to analyse forensic DNA samples and obtain massively parallel coverage of targeted short sequences with the variants they carry. We evaluated the levels of sequence coverage, genotyping precision, sensitivity and mixed DNA patterns of a prototype version of the first commercial forensic NGS kit: the HID-Ion AmpliSeq™ Identity Panel with 169-markers designed for the Ion PGM™ system. Evaluations were made between three laboratories following closely matched Ion PGM™ protocols and a simple validation framework of shared DNA controls. The sequence coverage obtained was extensive for the bulk of SNPs targeted by the HID-Ion AmpliSeq™ Identity Panel. Sensitivity studies showed 90–95% of SNP genotypes could be obtained from 25 to 100 pg of input DNA. Genotyping concordance tests included Coriell cell-line control DNA analyses checked against whole-genome sequencing data from 1000 Genomes and Complete Genomics, indicating a very high concordance rate of 99.8%. Discordant genotypes detected in rs1979255, rs1004357, rs938283, rs2032597 and rs2399332 indicate these loci should be excluded from the panel. Therefore, the HID-Ion AmpliSeq™ Identity Panel and Ion PGM™ system provide a sensitive and accurate forensic SNP genotyping assay. However, low-level DNA produced much more varied sequence coverage and in forensic use the Ion PGM™ system will require careful calibration of the total samples loaded per chip to preserve the genotyping reliability seen in routine forensic DNA. Furthermore, assessments of mixed DNA indicate the user’s control of sequence analysis parameter settings is necessary to ensure mixtures are detected robustly. Given the sensitivity of Ion PGM™, this aspect of forensic genotyping requires further optimisation before massively parallel sequencing is applied to routine casework.     

Cost-effectiveness and economic impact of the KineSpring® Knee Implant System in the treatment for knee osteoarthritis

Purpose

To investigate the cost-effectiveness and economic impact of the KineSpring System in the treatment for knee osteoarthritis in Germany.

Methods

Functional outcome scores of the general German population and knee osteoarthritis (OA) patients under surgical treatments (HTO, UKA and TKA), conservative treatments and treatment with the KineSpring System were used to derive the utility scores for each group. Quality-adjusted life years (QALYs) of each group were estimated using the utility scores. Finally, cost-utility analysis was performed using cost and QALYs data. The economic impact of knee OA in Germany was assessed in terms of annual total direct cost and indirect cost, total diseased population and potential QALYs saved with the KineSpring System.

Results

Assuming the durability of 10 years, the cost-utility ratio of the KineSpring System, surgical treatments and conservative treatments compared to no treatment in 2012 was €3,402/QALY, €4,899/QALY and €9,996/QALY, respectively. With even a lesser durability of 5 years, the cost-utility ratio of the KineSpring System maintained superiority over surgical treatments and conservative treatments (€7,327/QALY, €9,706/QALY and €10,467/QALY, respectively). The KineSpring System is a highly cost-effective alternative for knee osteoarthritis compared with the current accepted cost-effective threshold (willingness to pay) of $50,000 US/QALY gained. Our models suggest KineSpring System, if adapted widely could save up to 2.0 ± 0.07 million QALY assuming it has a 5-year durability and save up to 3.9 ± 0.1 million QALY assuming it has a 10-year durability.

Conclusion

An economic advantage for using the KineSpring System over other surgical and conservative treatments in knee OA patients in Germany is suggested by our model. According to currently accepted cost-effectiveness guidelines, the KineSpring Knee Implant System for knee OA is a cost-effective strategy.

Level of evidence

Cost-effectiveness analysis, Level III.     

Reliability of footprint geometric and plantar loading measurements in children using the Emed® M system

This study investigated the between-day reliability of footprint geometric and plantar loading measurements on children utilising the Emed^® M pressure measurement device. Bilateral footprints (static and dynamic) and foot loading measurements using the two-step gait method were collected on 21 children two days apart (age = 9.9 ± 1.8 years; mass = 34.6 ± 8.9 kg; height = 1.38 ± 0.12 m). Static and dynamic footprint geometric (lengths, widths and angles) and dynamic loading (pressures, forces, contact areas and contact time) parameters were compared. Intraclass correlation coefficients of static geometric parameters were varied (0.19–0.96), while superior results were achieved with dynamic geometric (0.66–0.98) and loading variables (0.52–0.94), with the exception of left contact time (0.37). Standard error of measurement recorded small absolute disparity for all geometric (length = 0.1–0.3 cm; arch index = 0.00–0.01; subarch angle = 0.6–6.2°; left/right foot progression angle = 0.5°/0.7°) and loading (peak pressure = 2.3–16.2 kPa; maximum force = 0.3–3.0%; total contact area = 0.28–0.49 cm²; % contact area = 0.1–0.6%; contact time = 32–79 ms) variables. Coefficient of variation displayed widest spread for static geometry (1.1–27.6%) followed by dynamic geometry (0.8–22.5%) and smallest spread for loading (1.3–16.8%) parameters. Limits of agreement (95%) were narrower in dynamic than static geometric parameters. Overall, the reliability of most dynamic geometric and loading parameters was good and excellent. Static electronic footprint measurements on children are not recommended due to their light body mass which results in incomplete footprints.     

Testing the Ion AmpliSeq™ HID Y-SNP Research Panel v1 for performance and resolution in admixed South Americans of haplogroup Q

Y haplogroups, defined by Y-SNPs, allow the reconstruction of the human Y chromosome genealogy, which is important for population, evolutionary and forensic genetics. In this study, Y-SNPs were typed and haplogroups inferred with the MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1, as a high-throughput approach. Firstly, the performance of the panel was evaluated with different DNA input amounts, reagent volumes and cycle numbers. DNA-inputs from 0.5 to 1 ng generated the most balanced read depth. Combined with full reagent and 19 cycles, this offered the highest number of amplicons with a sequencing read depth of at least 20 reads. Secondly, the sub-haplogroups of 182 admixed South Americans and Greenlanders belonging to haplogroup Q were inferred and tested for potential improvement in resolution. Most samples were assigned to lineage Q-M3 with some samples assigned to lineages upstream (Q-M346, L56, L57; Q-L331, L53; Q-L54; Q-CTS11969, CTS11970) or parallel (Q-L330, L334; Q-Z780/M971) to Q-M3. Only one sample was assigned to a downstream lineage (Q-Z35615, Z35616). Most individuals of haplogroup Q with NAM ancestry could neither be distinguished from each other, nor from half of the Greenlandic samples. Typing additional, known SNPs within lineage Q-M3, Z19483 and SA05, increased the resolution of predicted haplogroups. The search for novel variants in the sequenced regions allowed the detection of 42 variants and the subdivision of lineage Q-M3 into new subclades. The variants found in six of these subclades were exclusive to certain South American countries. In light of the limited differentiation of haplogroup Q samples, the additional information on known or novel SNPs disclosed in this study when using MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1 should be included in the Yleaf software, to increase the differentiation of lineage Q-M3.     

Massively parallel sequencing analysis of nondegraded and degraded DNA mixtures using the ForenSeq™ system in combination with EuroForMix software

Massively parallel sequencing (MPS) technologies enable the simultaneous analysis of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). MPS also enables the detection of alleles of the minor contributors in imbalanced DNA mixtures. In this study, 59 STRs (amelogenin, 27 autosomal STRs, 7 X-STRs, and 24 Y-STRs) and 94 identity-informative SNPs of 119 unrelated Taiwanese (50 men, 69 women) were sequenced using a commercial MPS kit. Forty-eight nondegraded and 44 highly degraded two-person artificial DNA mixtures with various minor to major ratios (1:9, 1:19, 1:29, 1:39, 1:79, and 1:99) were analyzed to examine the performance of this system for detecting the alleles of the minor contributors in DNA mixtures. Likelihood ratios based on continuous model were calculated using the EuroForMix for DNA mixture interpretation. The STR and SNP genotypes of these 119 Taiwanese were obtained. Several sequence variants of STRs were observed. Using EuroForMix software based on the sequence data of autosomal STRs and autosomal SNPs, 97.9% (47/48) and 97.7% (42/43) of minor donors were accurately inferred among the successfully analyzed nondegraded and degraded DNA mixtures, respectively. In conclusion, combined with EuroForMix software, this commercial kit is effective for assignment of the minor contributors in nondegraded and degraded DNA mixtures.

     

Low‐dose radiotherapy (LD‐RT) and the modulation of iNOS expression in adjuvant‐induced arthritis in rats

Purpose: Low‐dose radiotherapy (LD‐RT) of arthritic joints applied during the peak of the acute inflammatory response improves the clinical and histomorphological development of adjuvant arthritis. The study was undertaken to investigate the cellular composition of the inflammatory infiltrate and the expression of the pro‐inflammatory and anti‐inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclo‐oxygenase 2 (COX‐2) and haem‐oxygenase 1 (HO‐1), in response to LD‐RT.

Materials and methods: Adjuvant arthritis in female Lewis rats was induced by intradermal injection of heat‐inactivated mycobacterium tuberculosis on day 0. Both arthritic hind paws were sham irradiated (group 1) or X‐irradiated with either 5×1.0?Gy (group 2) or 5×0.5?Gy (group 3) from days 15 to 19 after induction (15 animals/group). On days 21 (n=12 joints/group) and 30 (n=18 joints/group), cryostat sections were analysed histologically and immunohistologically after specific staining for macrophages, iNOS, COX‐2 and HO‐1.

Results: A total of 5×1.0?Gy or 5×0.5?Gy led to a significant reduction of clinical symptoms from days 21 to 29, and a highly significant reduction of cartilage and bone destruction on day 30. Macrophage‐positive areas could be detected continuously throughout the periarticular infiltrate, and were slightly reduced after LD‐RT on days 21 and 30. This reduction was more pronounced after 5×1.0?Gy. Following LD‐RT, the iNOS score was reduced by about 45–50% on days 21 (p<0.05) and 30 (p<0.001). In contrast, the HO‐1 score was increased by about 50% on days 21 (p=0.08) and 30 (p=0.03).

Conclusions: The clinically and histologically observed prevention of the pro‐gression of adjuvant arthritis after LD‐RT given during the peak of the acute inflam‐matory response and the reduction of cartilage and bone destruction in the chronic phase appears to be related to the modulation of iNOS activity by low X‐ray doses.     

Evaluation of the Balance Error Scoring System (BESS) in the diagnosis of acute mountain sickness at 4380 m

Ascent to altitude is associated with a decrease in balance; however, the effect of acute mountain sickness (AMS) status on balance is variable depending on the test used and the altitude at which the test is performed. Here, we report preliminary findings on the relationship between the balance error scoring system (BESS) and AMS at the 2010 Janai Purnima festival at Gosainkunda, Nepal (4380?m). All subjects (n=37) completed a shortened BESS test (mBESS) while a subset completed the full BESS test (n=27). Pulse oximetry was used to measure heart rate and oxygen saturation, and blood pressure was measured by sphygmomanometer. Balance test scores (BESS and mBESS) and physiological measurements were compared between groups with AMS (AMS?) and without AMS (AMS?). Receiver-operator characteristic (ROC) curves were used to compare the abilities of the BESS and mBESS tests to correctly identify the AMS status of subjects. The AMS? group had significantly higher Lake Louise scores than the AMS? group (mean=4.0 (standard deviation=1.3) vs. 0.3 (0.6), p<0.001). The AMS? group also scored significantly higher on both the mBESS (6.6 (3.5) vs. 2.7 (1.7) errors, p=0.018) and the BESS tests (19.2 (8.8) vs. 10.4 (6.0) errors, p=0.001) compared to the AMS? group, indicating inferior balance in the AMS? group. The area under the ROC curve was significantly greater for the BESS test (0.895) compared to the mBESS test (0.690, p=0.02), implying that the full BESS test more accurately identified a subject's AMS status. Additional studies are needed to determine if BESS could be a useful adjunct to the clinical diagnosis of AMS.     

Population data and genetic characteristics of 12 X-STR loci using the Investigator® Argus X-12 Quality Sensor kit for the Kedayan population of Borneo in Malaysia

International Journal of Legal Medicine - DNA profiling of X-chromosomal short tandem repeats (X-STR) has exceptional value in criminal investigations, especially for complex kinship and incest...     

Genetic variation analysis of 15 autosomal STR loci of AmpFℓSTR® Sinofiler™ PCR Amplification Kit in Henan (central China) Han population

AmpF?STR® Sinofiler? PCR Amplification Kit is specially developed for Chinese forensic laboratories, but there are little population-genetic indices about this kit as a whole. This kit contains 15 STR loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D13S317, D16S539, D2S1338, D19S433, vWA, D18S51, D6S1043, D12S391, D5S818 and FGA. In order to evaluate this kit and to get basic population-genetic indices for its use in forensic practice in Chinese Han population, the DNA of 231 unrelated Han individuals from Henan (central China) were typed using the Kit. The most discriminating locus was D6S1043 while the least was D3S1358. The combined match probability was 9.81 × 10^?19 and the combined power of exclusion was 0.99999974. Statistical analysis of the generated data indicated no departure from expectation of Hardy–Weinberg Equilibrium (HWE) in all loci but D6S1043 and no linkage disequilibrium in all pairs of loci. The observed and expected heterozygosity, power of discrimination, polymorphic information content, the other population-genetic indices were calculated.     

Test–retest reliability of the Vestibular Ocular Motor Screening (VOMS) tool and modified Balance Error Scoring System (mBESS) in US military personnel

The Military Acute Concussion Evaluation 2 (MACE 2), which includes the Vestibular-Ocular Motor Screening (VOMS) tool and the single-leg stance component of the modified Balance Error Scoring System (mBESS), was introduced in 2018 as an assessment of acute mTBI in US military personnel. However, the reliability of the VOMS and mBESS in this population has not been established.ObjectivesThe primary purpose of this study was to examine the reliability of the VOMS across a 6-month period in healthy, uninjured US Army Special Operations Command (USASOC) personnel.DesignActive duty/heathy military personnel (n = 108) completed the VOMS and mBESS at baseline and follow-up 6 months later (±1 month).MethodCronbach's alpha was used to examine the internal consistency of the VOMS and mBESS at both time points. Two-way mixed intra-class correlation coefficients (ICC) with consistency agreement were used to evaluate test–retest reliability.ResultsVOMS demonstrated excellent internal consistency (α = 0.99), whereas, the mBESS demonstrated poor internal consistency (α = 0.29). Test–retest reliability of VOMS items was moderate-to-good with ICCs ranging from 0.60 to 0.81. Test–retest reliability was moderate for mBESS total score (ICC = 0.59) and double-leg stance (ICC = 0.73), while single-leg (ICC = 0.49) and tandem (ICC = 0.02) stances were poor.ConclusionsThe findings suggest that VOMS has high internal consistency and moderate-to-good test–retest reliability. mBESS has poor internal consistency and poor-to-moderate test–retest reliability. The results suggest that VOMS is a reliable addition to the MACE-2, whereas, mBESS single-leg stance is less reliable. As such, mBESS double-leg stance may be a more reliable measure of balance in this population.     

Allele frequencies of 31 autosomal short tandem repeat (auSTR) loci obtained using the Precision ID GlobalFiler™ NGS STR Panel v2 in 322 individuals from the Japanese population

In human identification methods that target short tandem repeats (STRs), massively parallel sequencing (MPS) technology has made it possible to genotype at the level of the specific sequence itself. This allows for the detection of repeat unit variants and single nucleotide polymorphisms (SNPs) adjacent to the STRs. Using the GlobalFiler™ NGS STR Panel v2, Ion S5, and Converge software, this study constructed a Japanese database of 31 autosomal STRs (auSTRs) and two sex markers from 322 individuals. After excluding some sequence errors and stutters, a total of 31 novel alleles were identified. Additionally, using the allele frequencies of 31 auSTR loci, the match probabilities for the length-based and sequence-based data were calculated to be 1.433 × 10⁻³⁴ and 9.163 × 10⁻³⁸, respectively. These values are at least nine orders of magnitude higher than that obtained from 21 auSTR loci in the Japanese population using the conventional capillary electrophoresis method. The database generated in this study is expected to be implemented in forensic practice and used to solve difficult casework.     

The Bücherer-Strecker synthesis of d- and l-(1-11C)tyrosine and the in vivo study of l-(1-11C)tyrosine in human brain using positron emission tomography

The synthesis of d-and l-(1-¹¹C)tyrosine, starting with ¹¹C-cyanide, is reported. dl-(1-¹¹C)Tyrosine was prepared by the Bücherer-Strecker reaction, from carrier added ¹¹C-cyanide with an incorporation of 80% in 20 min. The isolation of the pure d- and l-amino acid isomers from the enantiomeric mixture was accomplished within 15 min by preparative HPLC using a chiral stationary phase and a phosphate buffer as the mobile phase. Typically, the total synthesis time was 50 min (including purification) from end of trapping of ¹¹C-cyanide, with a radiochemical yield of d- and l-amino acid of 40%–60%. The d- and l-(1-¹¹C)tyrosine were both obtained optically pure, with a carrier added specific activity of 0.3–0.5 Ci/mmol and a radiochemical purity better than 99%. The ¹¹C labelled l-tyrosine was used in an in vivo study in the human brain using positron emission tomography (PET).     

Ionizing radiation modules of the expression and tyrosine phosphorylation of the focal adhesion‐associated proteins focal adhesion kinase (FAK) and its substrates p130cas and paxillin in A549 human lung carcinoma cells in vitro

Purpose: Focal adhesion kinase (FAK) is involved in the regulation of many cellular processes, including cell survival and death, proliferation and migration. The same endpoints are influenced by ionizing radiation (IR). Therefore, study was performed to determine the effect of IR on the expression and phosphorylation of FAK and two of its substrates, p130cas and paxillin, in vitro.

Materials and methods: Exponentially growing A549 lung carcinoma cells were exposed to 6?Gy X‐rays. Protein expression and the extent of tyrosine phosphorylation were investigated by immunoprecipitation experiments and Western blotting analysis using specific or unspecific phosphotyrosine antibodies. Immunofluorescence staining in combination with confocal laser scanning microscopy was done to localize the proteins within the cell.

Results: Tyrosine phosphorylation, of M_r 110?000–150?000 and 65?000–75?000 protein bands, was induced within 30?min after exposure to IR. Three of these proteins were identified as FAK, p130cas and paxillin. IR induced phosphorylation of FAK (tyr397 and tyr925) but did not change FAK expression. Additionally, IR induced phosphorylation of paxillin (tyr31 and tyr181) within 30?min and an up‐regulation of paxillin expression 2–6?h after exposure. Furthermore, a higher amount of phosphorylated p130cas was found in irradiated cells. Immunofluorescence staining demonstrated that in A549 cells, all three proteins colocalize at sites of focal adhesions at the cytoplasmic face of the cell membrane and to lamellopodia.

Conclusions: The data indicate that these focal adhesion‐associated proteins are modulated by IR and thus are likely to play a role in the cellular response to IR. These proteins might represent attractive targets to modulate FAK‐initiated signalling pathways, which may be involved in improved radioresistance and, furthermore, in important pathological phenomena such as tumour growth and metastatic phenotypes.