Forensic evaluation of the Asia Pacific ancestry-informative MAPlex assay.

DNA intelligence, and particularly the inference of biogeographical ancestry (BGA) is increasing in interest, and relevance within the forensic genetics community. The majority of current MPS-based forensic ancestry-informative assays focus on the differentiation of major global populations. The recently published MAPlex (Multiplex for the Asia Pacific) panel contains 144 SNPs and 20 microhaplotypes and aims to improve the differentiation of populations in the Asia Pacific region. This study reports the first forensic evaluation of the MAPlex panel using AmpliSeq technology and Ion S5 sequencing. This study reports on the overall performance of MAPlex including the assay's sequence coverage distribution and stability, baseline noise and description of problematic SNPs. Dilution series, artificially degraded and mixed DNA samples were also analysed to evaluate the sensitivity of the panel with challenging or compromised forensic samples. As the first panel to combine biallelic SNPs, multiple-allele SNPs and microhaplotypes, the MAPlex assay demonstrated an enhanced capacity for mixture detection, not easily performed with common binary SNPs. This performance evaluation indicates that MAPlex is a robust, stable and highly sensitive assay that is applicable to forensic casework for the prediction of BGA.

MAPlex - A massively parallel sequencing ancestry analysis multiplex for Asia-Pacific populations.

Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.

Inter-laboratory evaluation of the EUROFORGEN Global ancestry-informative SNP panel by massively parallel sequencing using the Ion PGM™.

The EUROFORGEN Global ancestry-informative SNP (AIM-SNPs) panel is a forensic multiplex of 128 markers designed to differentiate an individual's ancestry from amongst the five continental population groups of Africa, Europe, East Asia, Native America, and Oceania. A custom multiplex of AmpliSeq™ PCR primers was designed for the Global AIM-SNPs to perform massively parallel sequencing using the Ion PGM™ system. This study assessed individual SNP genotyping precision using the Ion PGM™, the forensic sensitivity of the multiplex using dilution series, degraded DNA plus simple mixtures, and the ancestry differentiation power of the final panel design, which required substitution of three original ancestry-informative SNPs with alternatives. Fourteen populations that had not been previously analyzed were genotyped using the custom multiplex and these studies allowed assessment of genotyping performance by comparison of data across five laboratories. Results indicate a low level of genotyping error can still occur from sequence misalignment caused by homopolymeric tracts close to the target SNP, despite careful scrutiny of candidate SNPs at the design stage. Such sequence misalignment required the exclusion of component SNP rs2080161 from the Global AIM-SNPs panel. However, the overall genotyping precision and sensitivity of this custom multiplex indicates the Ion PGM™ assay for the Global AIM-SNPs is highly suitable for forensic ancestry analysis with massively parallel sequencing.

Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise.

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.

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 100pg 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.

Building a forensic ancestry panel from the ground up: The EUROFORGEN Global AIM-SNP set.

Emerging next-generation sequencing technologies will enable DNA analyses to add pigmentation predictive and ancestry informative (AIM) SNPs to the range of markers detectable from a single PCR test. This prompted us to re-appraise current forensic and genomics AIM-SNPs and from the best sets, to identify the most divergent markers for a five population group differentiation of Africans, Europeans, East Asians, Native Americans and Oceanians by using our own online genome variation browsers. We prioritized careful balancing of population differentiation across the five group comparisons in order to minimize bias when estimating co-ancestry proportions in individuals with admixed ancestries. The differentiation of European from Middle East or South Asian ancestries was not chosen as a characteristic in order to concentrate on introducing Oceanian differentiation for the first time in a forensic AIM set. We describe a complete set of 128 AIM-SNPs that have near identical population-specific divergence across five continentally defined population groups. The full set can be systematically reduced in size, while preserving the most informative markers and the balance of population-specific divergence in at least four groups. We describe subsets of 88, 55, 28, 20 and 12 AIMs, enabling both new and existing SNP genotyping technologies to exploit the best markers identified for forensic ancestry analysis.

Glucocorticoid-regulated microRNAs and mirtrons in acute lymphoblastic leukemia.

Glucocorticoids (GCs) induce apoptosis in lymphoid lineage cells and are therefore used in the therapy of acute lymphoblastic leukemia (ALL) and related malignancies. MicroRNAs (miRNAs) and the related mirtrons are ~22 nucleotide RNAs derived from polymerase-II transcripts and implicated in the control of essential biological functions, including apoptosis. Whether GCs regulate miRNA-encoding transcription units is unknown. We investigated miRNA/mirtron expression and GC regulation in 8 leukemia/lymphoma in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time reverse transcription (RT)-PCR and northern blotting to detect mature miRNAs and/or their precursors. We found that mature miRNA regulations can be inferred from expression data of their host genes. Although a simple miRNA-initiated canonical pathway to GC-induced apoptosis or cell cycle arrest did not emerge, we identified several miRNAs/mirtrons that were regulated by GC in patients and cell lines, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing miR15 ~ 16 clusters. In an in vitro model, overexpression of miR15b ~ 16 mimics increased and silencing by miR15b ~ 16 inhibitors decreased GC sensitivity. Thus, the observed complex changes in miRNA/mirtron expression during GC treatment might contribute to the anti-leukemic GC effects in a cell context-dependent manner.

The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia.

Glucocorticoid (GC)-induced apoptosis is essential in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Pro- and anti-apoptotic members of the BCL2 family control many forms of apoptotic cell death, but the extent to which this survival 'rheostat' is involved in the beneficial effects of GC therapy is not understood. We performed systematic analyses of expression, GC regulation and function of BCL2 molecules in primary ALL lymphoblasts and a corresponding in vitro model. Affymetrix-based expression profiling revealed that the response included regulations of pro-apoptotic and, surprisingly, anti-apoptotic BCL2 family members, and varied among patients, but was dominated by induction of the BH3-only molecules BMF and BCL2L11/Bim and repression of PMAIP1/Noxa. Conditional lentiviral gene overexpression and knock-down by RNA interference in the CCRF-CEM model revealed that induction of Bim, and to a lesser extent that of BMF, was required and sufficient for apoptosis. Although anti-apoptotic BCL2 members were not regulated consistently by GC in the various systems, their overexpression delayed, whereas their knock-down accelerated, GC-induced cell death. Thus, the combined clinical and experimental data suggest that GCs induce both pro- and anti-apoptotic BCL2 family member-dependent pathways, with the outcome depending on cellular context and additional signals feeding into the BCL2 rheostat.