A collaborative study on the precision of the Markov chain Monte Carlo algorithms used for DNA profile interpretation.

Several fully continuous probabilistic genotyping software (PGS) use Markov chain Monte Carlo algorithms (MCMC) to assign weights to different proposed genotype combinations at a locus. Replicate interpretations of the same profile in these software are expected not to produce identical weights and likelihood ratio (LR) values due to the Monte Carlo aspect. This paper reports a detailed precision study under reproducibility conditions conducted as a collaborative exercise across the National Institute of Standards and Technology (NIST), Federal Bureau of Investigation (FBI), and Institute of Environmental Science and Research (ESR). Replicate interpretations generated across the three laboratories used the same input files, software version, and settings but different random number seed and different computers. This work demonstrates that using different computers to analyze replicate interpretations does not contribute to any variations in LR values. The study quantifies the magnitude of differences in the assigned LRs that is only due to run-to-run MCMC variability and addresses the potential explanations for the observed differences.

Sequence-based allelic variations and frequencies for 22 autosomal STR loci in the Lebanese population.

This is the first study that characterizes the sequence-based allelic variations of 22 autosomal Short Tandem Repeat (aSTR) loci in a population dataset collected from Lebanon. Genomic DNA extracts from 195 unrelated Lebanese individuals were amplified with PowerSeq 46GY System Prototype. Targeted amplicons were subjected to DNA library preparation and sequenced on the Verogen MiSeq FGx Sequencing System. Raw FASTQ data files were processed by STRait Razor v3. Sequence strings were annotated according to the considerations of the DNA Commission of the International Society for Forensic Genetics (ISFG) and tabulated herein with their respective allelic frequencies and GeneBank accession and version numbers. The sequenced Lebanese dataset resulted in 429 distinct allelic sequences as compared to the 236 alleles identified by length only. The increase in the number of alleles was observed at 18 out of 22 aSTR loci and was attributed to the sequence variations residing in both the STR repeat motifs and flanking regions. The study uncovered 25 novel aSTR allelic sequences across 12 loci for which GenBank records did not previously exist in the STRSeq BioProject, PRJNA380127. For a concordance check, the length-based allelic calls derived from the full sequences were compared to those genotyped using capillary electrophoresis (CE) methods. Population genetic parameters relevant to the evaluation of forensic DNA evidence were assessed for the sequence-based data and compared to the parameters generated from the length-based information. Using the sequence-based data, Analysis of MOlecular VAriance (AMOVA), genetic distances, and population genetic structure were evaluated for 1231 individuals sampled from the Lebanese and four U.S. populations (African American, Asian, Caucasian, and Hispanic). The results were tabulated and visualized in a population tree, multidimensional scaling scatter plots, and bar plots. This newly established sequence-based database for the Lebanese population can be beneficial for extending NGS applicability to casework or paternity testing and assessing the strength of evidence for NGS-STR profiles. The described novel sequence variants at certain loci can further help in the effort to characterize the sequence diversity of STR markers from different populations around the world.

Examining performance and likelihood ratios for two likelihood ratio systems using the PROVEDIt dataset.

A likelihood ratio (LR) system is defined as the entire pipeline of the measurement and interpretation processes where probabilistic genotyping software (PGS) is a piece of the whole LR system. To gain understanding on how two LR systems perform, a total of 154 two-person, 147 three-person, and 127 four-person mixture profiles of varying DNA quality, DNA quantity, and mixture ratios were obtained from the filtered (.CSV) files of the GlobalFiler 29 cycles 15s PROVEDIt dataset and deconvolved in two independently developed fully continuous programs, STRmix v2.6 and EuroForMix v2.1.0. Various parameters were set in each software and LR computations obtained from the two software were based on same/fixed EPG features, same pair of propositions, number of contributors, theta, and population allele frequencies. The ability of each LR system to discriminate between contributor (H1-true) and non-contributor (H2-true) scenarios was evaluated qualitatively and quantitatively. Differences in the numeric LR values and their corresponding verbal classifications between the two LR systems were compared. The magnitude of the differences in the assigned LRs and the potential explanations for the observed differences greater than or equal to 3 on the log10 scale were described. Cases of LR < 1 for H1-true tests and LR > 1 for H2-true tests were also discussed. Our intent is to demonstrate the value of using a publicly available ground truth known mixture dataset to assess discrimination performance of any LR system and show the steps used to understand similarities and differences between different LR systems. We share our observations with the forensic community and describe how examining more than one PGS with similar discrimination power can be beneficial, help analysts compare interpretation especially with low-template profiles or minor contributor cases, and be a potential additional diagnostic check even if software in use does contain certain diagnostic statistics as part of the output.

Understanding the characteristics of sequence-based single-source DNA profiles.

The sequencing of STR markers provides additional information present in the underlying sequence variation that is typically masked by traditional fragment-based genotyping. However, the interpretation of STR profiles generated by targeted sequencing methods are susceptible to the same factors encountered in profiles processed through capillary gel electrophoresis. These factors include stochastic variation, noise, stutter artifacts, heterozygote imbalance, and allelic drop-out/in. Our goal is to characterize and understand how these behave in targeted sequence datasets. Here, we developed a framework using statistical tools to systematically interpret the characteristics of single-source DNA profiles generated by targeted sequencing. Sensitivity studies were performed using known single-source samples amplified with the PowerSeq 46GY System Prototype with varying DNA target masses ranging from 15 pg to 500 pg. The STR loci were subjected to DNA library preparation using two commercially available library kits and sequenced on the Illumina MiSeq platform. Raw FASTQ data files were analyzed in STRait Razor v2.0 without applying any thresholds (at a coverage ≥ 1). We investigated the effect of library normalization on average locus coverage and studied methods for setting analytical and zygosity thresholds. All the data were analyzed per DNA quantity as well as investigated per method. Analyses presented can be applied to sequence data generated by similar targeted sequencing panels and/or NGS platforms.

Proximity Ligation Real-Time PCR: A protein-based confirmatory method for the identification of semen and sperm cells from sexual assault evidence.

The positive identification of seminal fluids in sexual assault crimes is considered crucial evidence to determine whether a sexual act occurred or not. However, current presumptive methods lack specificity and sensitivity. Confirmation of semen by microscopic examination of spermatozoa is laborious, time consuming, and can sometimes lead to negative or inconclusive results. Here we report the use of the Proximity Ligation Real-Time PCR (PLiRT-PCR) assay as an attractive and promising confirmatory method for the identification of semen and sperm proteins using two polyclonal antibodies, Prostate Specific Antigen (PSA) and Sperm-Specific Protein (SP10), respectively. PLiRT-PCR, relies on protein recognition by pairs of proximity probes (antibody-DNA conjugates) that give rise to a ligated DNA strand. The ligated DNA strand is then amplified and detected by qPCR.

Characterization of NIST human mitochondrial DNA SRM-2392 and SRM-2392-I standard reference materials by next generation sequencing.

Standard Reference Materials SRM 2392 and 2392-I are intended to provide quality control when amplifying and sequencing human mitochondrial genome sequences. The National Institute of Standards and Technology (NIST) offers these SRMs to laboratories performing DNA-based forensic human identification, molecular diagnosis of mitochondrial diseases, mutation detection, evolutionary anthropology, and genetic genealogy. The entire mtGenome (∼16569bp) of SRM 2392 and 2392-I have previously been characterized at NIST by Sanger sequencing. Herein, we used the sensitivity, specificity, and accuracy offered by next generation sequencing (NGS) to: (1) re-sequence the certified values of the SRM 2392 and 2392-I; (2) confirm Sanger data with a high coverage new sequencing technology; (3) detect lower level heteroplasmies (<20%); and thus (4) support mitochondrial sequencing communities in the adoption of NGS methods. To obtain a consensus sequence for the SRMs as well as identify and control any bias, sequencing was performed using two NGS platforms and data was analyzed using different bioinformatics pipelines. Our results confirm five low level heteroplasmy sites that were not previously observed with Sanger sequencing: three sites in the GM09947A template in SRM 2392 and two sites in the HL-60 template in SRM 2392-I.

The transcription factor YY1 is a novel substrate for Aurora B kinase at G2/M transition of the cell cycle.

Yin Yang 1 (YY1) is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. Polo-like kinase 1(Plk1) and Casein kinase 2α (CK2 α) were the first two kinases identified to phosphorylate YY1. In this study, we identify a third kinase. We report that YY1 is a novel substrate of the Aurora B kinase both in vitro and in vivo. Serine 184 phosphorylation of YY1 by Aurora B is cell cycle regulated and peaks at G2/M and is rapidly dephosphorylated, likely by protein phosphatase 1 (PP1) as the cells enter G1. Aurora A and Aurora C can also phosphorylate YY1 in vitro, but at serine/threonine residues other than serine 184. We present evidence that phosphorylation of YY1 in the central glycine/alanine (G/A)-rich region is important for DNA binding activity, with a potential phosphorylation/acetylation interplay regulating YY1 function. Given their importance in mitosis and overexpression in human cancers, Aurora kinases have been identified as promising therapeutic targets. Increasing our understanding of Aurora substrates will add to the understanding of their signaling pathways.

Phosphorylation of the transcription factor YY1 by CK2α prevents cleavage by caspase 7 during apoptosis.

In this report, we describe the phosphorylation of Yin Yang 1 (YY1) in vitro and in vivo by CK2α (casein kinase II), a multifunctional serine/threonine protein kinase. YY1 is a ubiquitously expressed multifunctional zinc finger transcription factor implicated in regulation of many cellular and viral genes. The products of these genes are associated with cell growth, the cell cycle, development, and differentiation. Numerous studies have linked YY1 to tumorigenesis and apoptosis. YY1 is a target for cleavage by caspases in vitro and in vivo as well, but very little is known about the mechanisms that regulate its cleavage during apoptosis. Here, we identify serine 118 in the transactivation domain of YY1 as the site of CK2α phosphorylation, proximal to a caspase 7 cleavage site. CK2α inhibitors, as well as knockdown of CK2α by small interfering RNA, reduce S118 phosphorylation in vivo and enhance YY1 cleavage under apoptotic conditions, whereas increased CK2α activity by overexpression in vivo elevates S118 phosphorylation. A serine-to-alanine substitution at serine 118 also increases the cleavage of YY1 during apoptosis compared to wild-type YY1. Taken together, we have discovered a regulatory link between YY1 phosphorylation at serine 118 and regulation of its cleavage during programmed cell death.

TNF-alpha modulates hepatic Na+-K+ ATPase activity via PGE2 and EP2 receptors.

The effect of TNF-alpha on liver Na(+)-K(+) ATPase was studied in Sprague-Dawley rats and in HepG2 cells. TNF-alpha was injected intraperitoneally to rats and 4h later the liver was isolated and the activity and protein expression of hepatic Na(+)-K(+) ATPase studied. The cytokine caused a significant down-regulation of the ATPase and a decrease in its activity. This effect disappeared in presence of indomethacin, an inhibitor of COX enzymes, and PGE2 injected to the animals imitated the effect of TNF-alpha. The observed in vivo effects of TNF and PGE2 on the pump appeared again when HepG2 cells were treated with the cytokine or the prostaglandin. The application of different agonist and antagonist to EP receptors showed that the effect of PGE2 is mediated via EP2 receptors. It was concluded that TNF-alpha induces in hepatocytes, PGE2 production which in turn reduces the activity and protein expression of the Na(+)-K(+) ATPase by activating EP2 receptors.