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The autosomal STR D6S474 and the Y-chromosomal STR DYS612 have been reported in multiple ways in the forensic literature, with differences in both the bracketed repeat structures and counting of numerical length-based capillary electrophoresis (CE) alleles. These issues often come to light when STR loci are introduced in commercial assays and results compared with historical publications of allele frequency data, or multiple assays are characterized with reference materials. We review the forensic literature and other relevant information, and provide suggestions for the future treatment of each STR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The de facto genetic markers of forensics are short tandem repeats (STRs). There are many analytical tools designed to work with STRs, including techniques for analyzing and assessing DNA mixtures. In contrast, the nascent field of forensic genetic genealogy often relies on biallelic single nucleotide polymorphisms (SNPs). Tools designed for the forensic assessment of SNPs are somewhat lacking, especially for DNA mixtures. In this paper we introduce Demixtify, a program that detects DNA mixtures using biallelic SNPs. Demixtify is quite powerful; highly imbalanced mixtures can be detected (≤1:99, considering in silico and in vitro mixtures) when coverage is ample. Demixtify can also detect mixtures in low coverage (∼1×) samples (when the mixture is relatively balanced). Demixtify includes an empirical estimator of sequence error that is specific to the markers assayed, making it especially relevant to the forensic community. Orthogonal techniques are also developed to characterize in vitro mixtures, as well as samples thought to be single source, and the results of these approaches serve to validate the techniques presented., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The genetic component of forensic genetic genealogy (FGG) is an estimate of kinship, often conducted at genome scales between a great number of individuals. The promise of FGG is substantial: in concert with genealogical records and other nongenetic information, it can indirectly identify a person of interest. A downside of FGG is cost, as it is currently expensive and requires chemistries uncommon to forensic genetic laboratories (microarrays and high throughput sequencing). The more common benchtop sequencers can be coupled with a targeted PCR assay to conduct FGG, though such approaches have limited resolution for kinship. This study evaluates low-pass sequencing, an alternative strategy that is accessible to benchtop sequencers and can produce resolutions comparable to high-pass sequencing. Samples from a three-generation pedigree were augmented to include up to 7th degree relatives (using whole genome pedigree simulations) and the ability to recover the true kinship coefficient was assessed using algorithms qualitatively similar to those found in GEDmatch. We show that up to 7th degree relatives can be reliably inferred from 1 × whole genome sequencing obtainable from desktop sequencers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The DNA Commission of the International Society for Forensic Genetics (ISFG) has developed a set of nomenclature recommendations for short tandem repeat (STR) sequences. These recommendations follow the 2016 considerations of the DNA Commission of the ISFG, incorporating the knowledge gained through research and population studies in the intervening years. While maintaining a focus on backward compatibility with the CE data that currently populate national DNA databases, this report also looks to the future with the establishment of recommended minimum sequence reporting ranges to facilitate interlaboratory comparisons, automated solutions for sequence-based allele designations, a suite of resources to support bioinformatic development, guidance for characterizing new STR loci, and considerations for incorporating STR sequences and other new markers into investigative databases., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bruce Budowle: Currently Consultant with Verogen., (Published by Elsevier B.V.)

PCR artifacts are an ever-present challenge in sequencing applications. These artifacts can seriously limit the analysis and interpretation of low-template samples and mixtures, especially with respect to a minor contributor. In medicine, molecular barcoding techniques have been employed to decrease the impact of PCR error and to allow the examination of low-abundance somatic variation. In principle, it should be possible to apply the same techniques to the forensic analysis of mixtures. To that end, several short tandem repeat loci were selected for targeted sequencing, and a bioinformatic pipeline for analyzing the sequence data was developed. The pipeline notes the relevant unique molecular identifiers (UMIs) attached to each read and, using machine learning, filters the noise products out of the set of potential alleles. To evaluate this pipeline, DNA from pairs of individuals were mixed at different ratios (1-1, 1-9) and sequenced with different starting amounts of DNA (10, 1 and 0.1 ng). Naïvely using the information in the molecular barcodes led to increased performance, with the machine learning resulting in an additional benefit. In concrete terms, using the UMI data results in less noise for a given amount of drop out. For instance, if thresholds are selected that filter out a quarter of the true alleles, using read counts accepts 2381 noise alleles and using raw UMI counts accepts 1726 noise alleles, while the machine learning approach only accepts 307., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Pancreaticoduodenectomy (PD) is complex procedure with high morbidity in the elderly. This retrospective study aimed to compare post-operative outcomes in patients ≥75 years of age who underwent robot-assisted (RA)PD and open PD., Methods: We analyzed 2502 patients ≥75 years of age who underwent PD from 2015 to 2018 in the National Surgical Quality Improvement Program (NSQIP) database. RAPD and open PD patients were propensity score matched 1:5 to assess the 30-day outcomes of interest: postoperative complications, length of stay, discharge destination, and readmissions., Results: Of 725 matched patients, 110 underwent RAPD, 615 OPD, and 12 were converted to an open operation. Post-operative outcomes were largely similar between cohorts. RAPD was associated a shorter length of stay (median 8 days, interquartile range [IQR] 6 to 11) than OPD (median 8 days, IQR 7 to 13) (p = 0.003). However, RAPD was associated with more readmissions (28.1% vs. 17.7%; p = 0.02)., Conclusions: RAPD in patients ≥75 years of age appears to be safe and has a similar complication profile to open PD. Randomized or well-designed prospective matched studies are needed to confirm these findings., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

One of the fundamental goals of forensic genetics is sample attribution, i.e., whether an item of evidence can be associated with some person or persons. The most common scenario involves a direct comparison, e.g., between DNA profiles from an evidentiary item and a sample collected from a person of interest. Less common is an indirect comparison in which kinship is used to potentially identify the source of the evidence. Because of the sheer amount of information lost in the hereditary process for comparison purposes, sampling a limited set of loci may not provide enough resolution to accurately resolve a relationship. Instead, whole genome techniques can sample the entirety of the genome or a sufficiently large portion of the genome and as such they may effect better relationship determinations. While relatively common in other areas of study, whole genome techniques have only begun to be explored in the forensic sciences. As such, bioinformatic pipelines are introduced for estimating kinship by massively parallel sequencing of whole genomes using approaches adapted from the medical and population genomic literature. The pipelines are designed to characterize a person's entire genome, not just some set of targeted markers. Two different variant callers are considered, contrasting a classical variant calling algorithm (BCFtools) to a more modern deep convolution neural network (DeepVariant). Two different bioinformatic pipelines specific to each variant caller are introduced and evaluated in a titration series. Filters and thresholds are then optimized specifically for the purposes of estimating kinship as determined by the KING-robust algorithm. With the appropriate filtering and thresholds in place both tools perform similarly, with DeepVariant tending to produce more accurate genotypes, though the resultant types of inaccuracies tended to produce slightly less accurate overall estimates of relatedness., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Short tandem repeats of the nuclear genome have been the preferred markers for analyzing forensic DNA mixtures. However, when nuclear DNA in a sample is degraded or limited, mitochondrial DNA (mtDNA) markers provide a powerful alternative. Though historically considered challenging, the interpretation and analysis of mtDNA mixtures have recently seen renewed interest with the advent of massively parallel sequencing. However, there are only a few software tools available for mtDNA mixture interpretation. To address this gap, the Mitochondrial Mixture Deconvolution and Interpretation Tool (MMDIT) was developed. MMDIT is an interactive application complete with a graphical user interface that allows users to deconvolve mtDNA (whole or partial genomes) mixtures into constituent donor haplotypes and estimate random match probabilities on these resultant haplotypes. In cases where deconvolution might not be feasible, the software allows mixture analysis directly within a binary framework (i.e. qualitatively, only using data on allele presence/absence). This paper explains the functionality of MMDIT, using an example of an in vitro two-person mtDNA mixture with a ratio of 1:4. The uniqueness of MMDIT lies in its ability to resolve mixtures into complete donor haplotypes using a statistical phasing framework before mixture analysis and evaluating statistical weights employing a novel graph algorithm approach. MMDIT is the first available open-source software that can automate mtDNA mixture deconvolution and analysis. The MMDIT web application can be accessed online at https://www.unthsc.edu/mmdit/. The source code is available at https://github.com/SammedMandape/MMDIT_UI and archived on zenodo (https://doi.org/10.5281/zenodo.4770184)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Forensic DNA typing typically relies on the length-based (LB) separation of PCR products containing short tandem repeat loci (STRs). Massively parallel sequencing (MPS) elucidates an additional level of STR motif and flanking region variation. Also, MPS enables simultaneous analysis of different marker-types - autosomal STRs, SNPs for lineage and identification purposes, reducing both the amount of sample used and the turn-around-time of analysis. Therefore, MPS methodologies are being considered as an additional tool in forensic genetic casework. The PowerSeq™ Auto/Y System (Promega Corp), a multiplex forensic kit for MPS, enables analysis of the 22 autosomal STR markers (plus Amelogenin) from the PowerPlex® Fusion 6C kit and 23 Y-STR markers from the PowerPlex® Y23 kit. Population data were generated from 140 individuals from an admixed sample from Rio de Janeiro, Brazil. All samples were processed according to the manufacturers' recommended protocols. Raw data (FastQ) were generated for each indexed sample and analyzed using STRait Razor v2s and PowerSeqv2.config file. The subsequent population data showed the largest increase in expected heterozygosity (23%), from LB to sequence-based (SB) analyses at the D5S818 locus. Unreported allele was found at the D21S11 locus. The random match probability across all loci decreased from 5.9 × 10 -28 to 7.6 × 10 -33 . Sensitivity studies using 1, 0.25, 0.062 and 0.016 ng of DNA input were analyzed in triplicate. Full Y-STR profiles were detected in all samples, and no autosomal allele drop-out was observed with 62 pg of input DNA. For mixture studies, 1 ng of genomic DNA from a male and female sample at 1:1, 1:4, 1:9, 1:19 and 1:49 proportions were analyzed in triplicate. Clearly resolvable alleles (i.e., no stacking or shared alleles) were obtained at a 1:19 male to female contributor ratio. The minus one stutter (-1) increased with the longest uninterrupted stretch (LUS) allele size reads and according to simple or compound/complex repeats. The haplotype-specific stutter rates add more information for mixed samples interpretation. These data support the use of the PowerSeq TM Auto/Y systems prototype kit (22 autosomal STR loci, 23 Y-STR loci and Amelogenin) for forensic genetics applications., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Since 2013, STRait Razor has enabled analysis of massively parallel sequencing (MPS) data from various marker systems such as short tandem repeats, single nucleotide polymorphisms, insertion/deletions, and mitochondrial DNA. In this paper, STRait Razor Online (SRO), available at https://www.unthsc.edu/straitrazor, is introduced as an interactive, Shiny-based user interface for primary analysis of MPS data and secondary analysis of STRait Razor haplotype pileups. This software can be accessed from any common browser via desktop, tablet, or smartphone device. SRO is available also as a standalone application and open-source R script available at https://github.com/ExpectationsManaged/STRaitRazorOnline. The local application is capable of batch processing of both fastq files and primary analysis output. Processed batches generate individual report folders and summary reports at the locus- and haplotype-level in a matter of minutes. For example, the processing of data from ∼700 samples generated with the ForenSeq Signature Preparation Kit from allsequences.txt to a final table can be performed in ∼40 min whereas the Excel-based workbooks can take 35-60 h to compile a subset of the tables generated by SRO. To facilitate analysis of single-source, reference samples, a preliminary triaging system was implemented that calls potential alleles and flags loci suspected of severe heterozygote imbalance. When compared to published, manually curated data sets, 98.72 % of software-assigned allele calls without manual interpretation were consistent with curated data sets, 0.99 % loci were presented to the user for interpretation due to heterozygote imbalance, and the remaining 0.29 % of loci were inconsistent due to the analytical thresholds used across the studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Autosomal DNA data from Peru for human identity testing purposes are scarce in the scientific literature, which hinders obtaining an appropriate portrait of the genetic variation of the resident populations. In this study we genetically characterize five populations from the Northeastern Peruvian Andes (Chachapoyas, Awajún, Wampís, Huancas and Cajamarca). Autosomal short tandem repeat (aSTR) and identity informative single nucleotide polymorphism (iiSNP) data from a total of 233 unrelated individuals are provided, and forensic genetic parameters are calculated for each population and for the combined set Northeastern Peruvian Andes. After correction for multiple testing in the whole dataset of the Northeastern Peruvian Andes, the only departure from Hardy-Weinberg equilibrium was observed in locus rs2111980. Twenty one out of 27 aSTR loci exhibited an increased number of alleles due to sequence variation in the repeat motif and flanking regions. For iiSNPs 33% of the loci displayed flanking region variation. The combined random match probability (RMP), assuming independence of all loci (aSTRs and iiSNPs), in the Chachapoyas, the population with the largest samples size (N = 172), was 8.14 × 10 -62 for length-based data while for sequence-based was 4.15 × 10 -67 . In the merged dataset (Northeastern Peruvian Andes; N = 233), the combined RMP when including all markers were 2.96 × 10 -61 (length-based) and 3.21 × 10 -66 (sequence-based). These new data help to fill up some of the gaps in the genetic canvas of South America and provide essential length- and sequence-based background information for other forensic genetic studies in Peru., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Unique molecular identifiers (UMIs) are a promising approach to contend with errors generated during PCR and massively parallel sequencing (MPS). With UMI technology, random molecular barcodes are ligated to template DNA molecules prior to PCR, allowing PCR and sequencing error to be tracked and corrected bioinformatically. UMIs have the potential to be particularly informative for the interpretation of short tandem repeats (STRs). Traditional MPS approaches may simply lead to the observation of alleles that are consistent with the hypotheses of stutter, while with UMIs stutter products bioinformatically may be re-associated with their parental alleles and subsequently removed. Herein, a bioinformatics pipeline named strumi is described that is designed for the analysis of STRs that are tagged with UMIs. Unlike other tools, strumi is an alignment-free machine learning driven algorithm that clusters individual MPS reads into UMI families, infers consensus super-reads that represent each family and provides an estimate the resulting haplotype's accuracy. Super-reads, in turn, approximate independent measurements not of the PCR products, but of the original template molecules, both in terms of quantity and sequence identity. Provisional assessments show that naïve threshold-based approaches generate super-reads that are accurate (∼97 % haplotype accuracy, compared to ∼78 % when UMIs are not used), and the application of a more nuanced machine learning approach increases the accuracy to ∼99.5 % depending on the level of certainty desired. With these features, UMIs may greatly simplify probabilistic genotyping systems and reduce uncertainty. However, the ability to interpret alleles at trace levels also permits the interpretation, characterization and quantification of contamination as well as somatic variation (including somatic stutter), which may present newfound challenges., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Reverse Complement PCR (RC-PCR) is an innovative, one-step PCR target enrichment technology adapted for the amplification of highly degraded (fragmented) DNA. It provides simultaneous amplification and tagging of a targeted sequence construct in a single, closed-tube assay. A human identification (HID) RC-PCR panel was designed targeting 27 identity single nucleotide polymorphisms (SNPs) generating targets only 50 base pairs in length. In a single reaction, the complete sequencing construct is produced which is essential for massively parallel sequencing (MPS) library preparation, thus reducing time and labor as well as minimizing the risk of sample carry-over or other forms of contamination. The RC-PCR system was evaluated and found to produce reliable and concordant variant calls. Also, the RC-PCR system demonstrated to have substantial sensitivity of detection with a majority of alleles detected at 60 pg of input DNA and robustness in tolerating known PCR inhibitors. The RC-PCR system may be an effective alternative to current forensic genetic methods in the analysis of highly degraded DNA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Compound repeats provide a testing ground for evaluating hypotheses on the causes and consequences of stutter. As compound repeats have two repeating motifs, each motif may produce stutter variants; thus as the repeat within the locus is compound, so is the resultant stutter. Further, the rates of stutter formation between these motifs may not be independent. This lack of independence may complicate modeling strategies, thus contributing to the challenges of mixture interpretation that rely on nucleotide sequence. This study evaluates compound stutter in two STR loci: D2S1338 and D12S391. The effects of flanking variation, as well as possible interactions between the two different uninterrupted stretches (US) and their respective stutter variants are assessed. Multivariate multiple linear regression (MMLR) was used to show that, as with simple repeats, the rate of stutter product formation of a particular repeating motif is not solely a function of the US of that repeat. The nucleotides adjacent to the repeating motif also appear to influence the rate of stutter formation of that motif, with those nucleotides sometimes including the other motif. MMLR was used to estimate the size of these effects and to construct an example of a two-dimensional (thus, a compound) stutter prediction. This example may merit further investigation in the application of massively parallel sequencing data to mixture interpretation and probabilistic genotyping., (Copyright © 2018. Published by Elsevier B.V.)

Cyanobacteria are photosynthetic organisms responsible for ~25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme in the biosphere. Applying Zernike phase-contrast electron cryo-tomography and automated annotation, we identified individual RuBisCO molecules and their assembly intermediates leading to the formation of carboxysomes inside Syn5 cyanophage infected cyanobacteria Synechococcus sp. WH8109 cells. Surprisingly, more RuBisCO molecules were found to be present as cytosolic free-standing complexes or clusters than as packaged assemblies inside carboxysomes. Cytosolic RuBisCO clusters and partially assembled carboxysomes identified in the cell tomograms support a concurrent assembly model involving both the protein shell and the enclosed RuBisCO. In mature carboxysomes, RuBisCO is neither randomly nor strictly icosahedrally packed within protein shells of variable sizes. A time-averaged molecular dynamics simulation showed a semi-liquid probability distribution of the RuBisCO in carboxysomes and correlated well with carboxysome subtomogram averages. Our structural observations reveal the various stages of RuBisCO assemblies, which could be important for understanding cellular function., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Sequencing whole mitochondrial genomes by capillary electrophoresis is a costly and time/labor-intensive endeavor. Many of the previous Sanger sequencing-based approaches generated amplicons that were several kilobases in length; lengths that are likely not amenable for most forensic applications. However, with the advent of massively parallel sequencing (MPS) short-amplicon multiplexes covering the entire mitochondrial genome can be sequenced relatively easily and rapidly. Recently, the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific by Applied Biosystems™) has been introduced. This panel is composed of 162 amplicons (in two multiplexes) that are considerably smaller in length (∼163bp) and thus are more amenable to analyzing challenged samples. This panel was evaluated on both the Ion S5™ System (Thermo Fisher Scientific) and the MiSeq™ FGx Desktop Sequencer (Illumina). A script was developed to extract phased haplotypes associated with these amplicons. Levels of read-depth were compared across sequencing pools and between sequencing technologies and haplotype concordances were assessed. Given modest thresholds on read depth, the haplotypes identified by either technology were consistent. Nuclear mitochondrial sequences (Numts) were also inferred, and the effect of different mapping strategies commonly used to filter out Numts were contrasted. Some Numts are co-amplified with this amplification kit, and while the choice of reference sequence can mitigate some of these effects, some data from the mitochondrial genome were lost in the process in this study. This study demonstrates that the Ion and MiSeq platforms provide consistent haplotype estimation of the whole mitochondrial genome, thus providing further support for the reliability and validity of the Precision ID mtDNA Whole Genome Panel., (Copyright © 2018. Published by Elsevier B.V.)

The use of single nucleotide polymorphisms (SNPs) in forensic genetics has been limited to challenged samples with low template and/or degraded DNA. The recent introduction of massively parallel sequencing (MPS) technologies has expanded the potential applications of these markers and increased the discrimination power of well-established loci by considering variation in the flanking regions of target loci. The ForenSeq Signature Preparation Kit contains 165 SNP amplicons for ancestry- (aiSNPs), identity- (iiSNPs), and phenotype-inference (piSNPs). In this study, 714 individuals from four major populations (African American, AFA; East Asian, ASN; US Caucasian, CAU; and Southwest US Hispanic, HIS) previously reported by Churchill et al. [Forensic Sci Int Genet. 30 (2017) 81-92; DOI: https://doi.org/10.1016/j.fsigen.2017.06.004] were assessed using STRait Razor v2s to determine the level of diversity in the flanking regions of these amplicons. The results show that nearly 70% of loci showed some level of flanking region variation with 22 iiSNPs and 8 aiSNPs categorized as microhaplotypes in this study. The heterozygosities of these microhaplotypes approached, and in one instance surpassed, those of some core STR loci. Also, the impact of the flanking region on other forensic parameters (e.g., power of exclusion and power of discrimination) was examined. Sixteen of the 94 iiSNPs had an effective allele number greater than 2.00 across the four populations. To assess what effect the flanking region information had on the ancestry inference, genotype probabilities and likelihood ratios were determined. Additionally, concordance with the ForenSeq UAS and Nextera Rapid Capture was evaluated, and patterns of heterozygote imbalance were identified. Pairwise comparison of the iiSNP diplotypes determined the probability of detecting a mixture (i.e., observing ≥ 3 haplotypes) using these loci alone was 0.9952. The improvement in random match probabilities for the full regions over the target iiSNPs was found to be significant. When combining the iiSNPs with the autosomal STRs, the combined match probabilities ranged from 6.40 × 10 -73 (ASN) to 1.02 × 10 -79 (AFA)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Pancreatic fistula is a major cause of morbidity after pancreas surgery. In 2014, a single-center, randomized-controlled trial found pasireotide decreased pancreatic fistula rates. However, this finding has not been validated, nor has pasireotide been widely adopted., Methods: A single-arm study in 111 consecutive patients undergoing pancreatic resection April 2015-October 2016 was conducted. Beginning immediately before surgery, patients received 900 μg subcutaneous pasireotide twice daily for up to seven days. Fistula rates were compared to 168 historical controls from July 2013 to March 2015. The primary outcome was Grade B/C fistula, as defined by the International Study Group on Pancreatic Fistula (ISGPF)., Results: There were no significant differences between the pasireotide group and historical controls in demographics, comorbidities, operation type, malignancy, gland texture, or pancreatic duct size. Pasireotide did not reduce fistula rate (15.5% control versus 17.1% pasireotide, p = 0.72). In subgroup analyses of pancreaticoduodenectomy or distal pancreatectomy, or patients with soft gland texture and/or small duct size, there was no decrease in fistulas. Thirty-nine patients (38%) experienced dose-limiting nausea., Conclusions: In an appropriately-powered, single-institution prospective study, pasireotide was not validated as a preventive measure for pancreatic fistula., (Copyright © 2017 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

The human skin microbiome is comprised of diverse communities of bacterial, eukaryotic, and viral taxa and contributes millions of additional genes to the repertoire of human genes, affecting human metabolism and immune response. Numerous genetic and environmental factors influence the microbiome composition and as such contribute to individual-specific microbial signatures which may be exploited for forensic applications. Previous studies have demonstrated the potential to associate skin microbial profiles collected from touched items to their individual owner, mainly using unsupervised methods from samples collected over short time intervals. Those studies utilize either targeted 16S rRNA or shotgun metagenomic sequencing to characterize skin microbiomes; however, these approaches have limited species and strain resolution and susceptibility to stochastic effects, respectively. Clade-specific markers from the skin microbiome, using supervised learning, can predict individual identity using skin microbiomes from their respective donors with high accuracy. In this study the hidSkinPlex is presented, a novel targeted sequencing method using skin microbiome markers developed for human identification. The hidSkinPlex (comprised of 286 bacterial (and phage) family-, genus-, species-, and subspecies-level markers), initially was evaluated on three bacterial control samples represented in the panel (i.e., Propionibacterium acnes, Propionibacterium granulosum, and Rothia dentocariosa) to assess the performance of the multiplex. The hidSkinPlex was further evaluated for prediction purposes. The hidSkinPlex markers were used to attribute skin microbiomes collected from eight individuals from three body sites (i.e., foot (Fb), hand (Hp) and manubrium (Mb)) to their host donor. Supervised learning, specifically regularized multinomial logistic regression and 1-nearest-neighbor classification were used to classify skin microbiomes to their hosts with up to 92% (Fb), 96% (Mb), and 100% (Hp) accuracy. All samples (n=72) regardless of body site origin were correctly classified with up to 94% accuracy, and body site origin could be predicted with up to 86% accuracy. Finally, human short tandem repeat and single-nucleotide polymorphism profiles were generated from skin swab extracts from a single subject to highlight the potential to use microbiome profiling in conjunction with low-biomass samples. The hidSkinPlex is a novel targeted enrichment approach to profile skin microbiomes for human forensic identification purposes and provides a method to further characterize the utility of skin microflora for human identification in future studies, such as the stability and diversity of the personal skin microbiome., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The STR Sequencing Project (STRSeq) was initiated to facilitate the description of sequence-based alleles at the Short Tandem Repeat (STR) loci targeted in human identification assays. This international collaborative effort, which has been endorsed by the ISFG DNA Commission, provides a framework for communication among laboratories. The initial data used to populate the project are the aggregate alleles observed in targeted sequencing studies across four laboratories: National Institute of Standards and Technology (N=1786), Kings College London (N=1043), University of North Texas Health Sciences Center (N=839), and University of Santiago de Compostela (N=944), for a total of 4612 individuals. STRSeq data are maintained as GenBank records at the U.S. National Center for Biotechnology Information (NCBI), which participates in a daily data exchange with the DNA DataBank of Japan (DDBJ) and the European Nucleotide Archive (ENA). Each GenBank record contains the observed sequence of a STR region, annotation ("bracketing") of the repeat region and flanking region polymorphisms, information regarding the sequencing assay and data quality, and backward compatible length-based allele designation. STRSeq GenBank records are organized within a BioProject at NCBI (https://www.ncbi.nlm.nih.gov/bioproject/380127), which is sub-divided into: commonly used autosomal STRs, alternate autosomal STRs, Y-chromosomal STRs, and X-chromosomal STRs. Each of these categories is further divided into locus-specific BioProjects. The BioProject hierarchy facilitates access to the GenBank records by browsing, BLAST searching, or ftp download. Future plans include user interface tools at strseq.nist.gov, a pathway for submission of additional allele records by laboratories performing population sample sequencing and interaction with the STRidER web portal for quality control (http://strider.online)., (Published by Elsevier B.V.)

The short tandem repeat allele identification tool (STRait Razor), a program used to characterize the haplotypes of short tandem repeats (STRs) in massively parallel sequencing (MPS) data, was redesigned. STRait Razor v3.0 performs ∼660× faster allele identification than its previous version (v2s), a speedup that is largely due to a novel indexing strategy used to perform "fuzzy" (approximate) string matching of anchor sequences. Written in a portable compiled language, C++, STRait Razor v3.0 functions on all major operating systems including Microsoft Windows, and it has cross-platform multithreading support. In silico estimates of precision and accuracy of STRait Razor v3.0 were 100% in this evaluation and results were highly concordant with those of Strait Razor v2s. STRait Razor v3.0 adds several key features that simplify the haplotype reporting process, including simple filters to remove low frequency haplotypes as well as merging haplotypes within a locus encoded on opposite strands of the DNA molecule., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The MiSeq FGx Forensic Genomics System (Illumina) enables amplification and massively parallel sequencing of 59 STRs, 94 identity informative SNPs, 54 ancestry informative SNPs, and 24 phenotypic informative SNPs. Allele frequency and population statistics data were generated for the 172 SNP loci included in this panel on four major population groups (Chinese, African Americans, US Caucasians, and Southwest Hispanics). Single-locus and combined random match probability values were generated for the identity informative SNPs. The average combined STR and identity informative SNP random match probabilities (assuming independence) across all four populations were 1.75E-67 and 2.30E-71 with length-based and sequence-based STR alleles, respectively. Ancestry and phenotype predictions were obtained using the ForenSeq™ Universal Analysis System (UAS; Illumina) based on the ancestry informative and phenotype informative SNP profiles generated for each sample. Additionally, performance metrics, including profile completeness, read depth, relative locus performance, and allele coverage ratios, were evaluated and detailed for the 725 samples included in this study. While some genetic markers included in this panel performed notably better than others, performance across populations was generally consistent. The performance and population data included in this study support that accurate and reliable profiles were generated and provide valuable background information for laboratories considering internal validation studies and implementation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

STRait Razor has provided the forensic community a free-to-use, open-source tool for short tandem repeat (STR) analysis of massively parallel sequencing (MPS) data. STRait Razor v2s (SRv2s) allows users to capture physically phased haplotypes within the full amplicon of both commercial (ForenSeq) and "early access" panels (PowerSeq, Mixture ID). STRait Razor v2s may be run in batch mode to facilitate population-level analysis and is supported by all Unix distributions (including MAC OS). Data are reported in tables in string (haplotype), length-based (e.g., vWA allele 14), and International Society of Forensic Genetics (ISFG)-recommended (vWA [CE 14]-GRCh38-chr12:5983950-5984049 (TAGA) 10 (CAGA) 3 TAGA) formats. STRait Razor v2s currently contains a database of ∼2500 unique sequences. This database is used by SRv2s to match strings to the appropriate allele in ISFG-recommended format. In addition to STRs, SRv2s has configuration files necessary to capture and report haplotypes from all marker types included in these multiplexes (e.g., SNPs, InDels, and microhaplotypes). To facilitate mixture interpretation, data may be displayed from all markers in a format similar to that of electropherograms displayed by traditional forensic software. The download package for SRv2s may be found at https://www.unthsc.edu/graduate-school-of-biomedical-sciences/molecular-and-medical-genetics/laboratory-faculty-and-staff/strait-razor., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We report here a novel multiplexed DNA analysis system consisting of 20 Alu markers and Amelogenin for analysis of highly degraded forensic biological samples. The key to the success of the system in obtaining results from degraded samples is the primer design yielding small amplicon size (60-125bp) for all 20 markers. The markers included in the InnoTyper ® 21 system are bi-allelic, having two possible allelic states (insertion or null) and thus termed INNULs. The markers are short interspersed nuclear elements (SINEs), a category of retrotransposable elements (REs) which are non-coding genomic DNA repeat sequences, or "mobile insertion elements," comprising approximately 40% of the human genome. Alu elements are primate specific SINEs that have reached a copy number in excess of one million in the human genome, which makes these markers highly sensitive and desirable for forensic samples with extremely degraded DNA. Until now however, due to the inherent size difference associated with insertion and no insertion alleles, the use of Alu REs has not been practical for forensic applications. The novel primer design described herein has allowed the development of a multiplexed Alu system yielding fragment sizes amenable to degraded DNA samples, as frequently encountered in missing persons cases or forensic samples such as hair shafts. Although use of Alus in human identity has been studied using single marker amplification and reported before, we report for the first time development and validation of a system with multiplexed RE markers. Studies performed include PCR optimization, species specificity, sensitivity, degradation and inhibition, precision and accuracy, nonprobative samples, mixture, and population database studies. A population study using 592 samples including five populations was performed using InnoTyper 21. The data indicated the random match probability for the combination of these 20 Alu markers was greater than 1 in 3.8 million for the populations studied, indicating the greater statistical power of these autosomal nuclear DNA markers over haplotype systems typically used in such degraded samples. Results demonstrate the system is successful in obtaining results from highly degraded DNA. A sensitivity study performed demonstrated at least 95% recovery of alleles from as low as 50pg of total input DNA, and partial profiles from as low as 25pg. This study has demonstrated that the bi-allelic INNULs in the InnoTyper 21 system provide a sensitivity of detection and a power of discrimination that makes them useful for human identification of extremely degraded samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Massively parallel sequencing (MPS) offers advantages over current capillary electrophoresis-based analysis of short tandem repeat (STR) loci for human identification testing. In particular STR repeat motif sequence information can be obtained, thereby increasing the discrimination power of some loci. While sequence variation within the repeat region is observed relatively frequently in some of the commonly used STRs, there is an additional degree of variation found in the flanking regions adjacent to the repeat motif. Repeat motif and flanking region sequence variation have been described for major population groups, however, not for more isolated populations. Flanking region sequence variation in STR and single nucleotide polymorphism (SNP) loci in the Yavapai population was analyzed using the ForenSeq™ DNA Signature Prep Kit and STRait Razor v2s. Seven and 14 autosomal STRs and identity-informative single nucleotide polymorphisms (iiSNPs), respectively, had some degree of flanking region variation. Three and four of these identity-informative loci, respectively, showed ≥5% increase in expected heterozygosity. The combined length- and sequence-based random match probabilities (RMPs) for 27 autosomal STRs were 6.11×10 -26 and 2.79×10 -29 , respectively. When combined with 94 iiSNPs (a subset of which became microhaplotypes) the combined RMP was 5.49×10 -63 . Analysis of length-based and sequence-based autosomal STRs in STRUCTURE indicated that the Yavapai are most similar to the Hispanic population. While producing minimal increase in X- and Y-STR discrimination potential, access to flanking region data enabled identification of one novel X-STR and three Y-STR alleles relative to previous reports. Five ancestry-informative SNPs (aiSNPs) and two phenotype-informative SNPs (piSNPs) exhibited notable flanking region variation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Massively parallel sequencing (MPS) can identify sequence variation within short tandem repeat (STR) alleles as well as their nominal allele lengths that traditionally have been obtained by capillary electrophoresis. Using the MiSeq FGx Forensic Genomics System (Illumina), STRait Razor, and in-house excel workbooks, genetic variation was characterized within STR repeat and flanking regions of 27 autosomal, 7 X-chromosome and 24 Y-chromosome STR markers in 777 unrelated individuals from four population groups. Seven hundred and forty six autosomal, 227 X-chromosome, and 324 Y-chromosome STR alleles were identified by sequence compared with 357 autosomal, 107 X-chromosome, and 189 Y-chromosome STR alleles that were identified by length. Within the observed sequence variation, 227 autosomal, 156 X-chromosome, and 112 Y-chromosome novel alleles were identified and described. One hundred and seventy six autosomal, 123 X-chromosome, and 93 Y-chromosome sequence variants resided within STR repeat regions, and 86 autosomal, 39 X-chromosome, and 20 Y-chromosome variants were located in STR flanking regions. Three markers, D18S51, DXS10135, and DYS385a-b had 1, 4, and 1 alleles, respectively, which contained both a novel repeat region variant and a flanking sequence variant in the same nucleotide sequence. There were 50 markers that demonstrated a relative increase in diversity with the variant sequence alleles compared with those of traditional nominal length alleles. These population data illustrate the genetic variation that exists in the commonly used STR markers in the selected population samples and provide allele frequencies for statistical calculations related to STR profiling with MPS data., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Short tandem repeat (STR) loci are the traditional markers used for kinship, missing persons, and direct comparison human identity testing. These markers hold considerable value due to their highly polymorphic nature, amplicon size, and ability to be multiplexed. However, many STRs are still too large for use in analysis of highly degraded DNA. Small bi-allelic polymorphisms, such as insertions/deletions (INDELs), may be better suited for analyzing compromised samples, and their allele size differences are amenable to analysis by capillary electrophoresis. The INDEL marker allelic states range in size from 2 to 6 base pairs, enabling small amplicon size. In addition, heterozygote balance may be increased by minimizing preferential amplification of the smaller allele, as is more common with STR markers. Multiplexing a large number of INDELs allows for generating panels with high discrimination power. The Nextera™ Rapid Capture Custom Enrichment Kit (Illumina, Inc., San Diego, CA) and massively parallel sequencing (MPS) on the Illumina MiSeq were used to sequence 68 well-characterized INDELs in four major US population groups. In addition, the STR Allele Identification Tool: Razor (STRait Razor) was used in a novel way to analyze INDEL sequences and detect adjacent single nucleotide polymorphisms (SNPs) and other polymorphisms. This application enabled the discovery of unique allelic variants, which increased the discrimination power and decreased the single-locus random match probabilities (RMPs) of 22 of these well-characterized INDELs which can be considered as microhaplotypes. These findings suggest that additional microhaplotypes containing human identification (HID) INDELs may exist elsewhere in the genome., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Forensically-relevant genetic markers were typed for sixty-two Yavapai Native Americans using the ForenSeq™ DNA Signature Prep Kit.These data are invaluable to the human identity community due to the greater genetic differentiation among Native American tribes than among other subdivisions within major populations of the United States. Autosomal, X-chromosomal, and Y-chromosomal short tandem repeat (STR) and identity-informative (iSNPs), ancestry-informative (aSNPs), and phenotype-informative (pSNPs) single nucleotide polymorphism (SNP) allele frequencies are reported. Sequence-based allelic variants were observed in 13 autosomal, 3 X, and 3 Y STRs. These observations increased observed and expected heterozygosities for autosomal STRs by 0.081±0.068 and 0.073±0.063, respectively, and decreased single-locus random match probabilities by 0.051±0.043 for 13 autosomal STRs. The autosomal random match probabilities (RMPs) were 2.37×10-26 and 2.81×10-29 for length-based and sequence-based alleles, respectively. There were 22 and 25 unique Y-STR haplotypes among 26 males, generating haplotype diversities of 0.95 and 0.96, for length-based and sequencebased alleles, respectively. Of the 26 haplotypes generated, 17 were assigned to haplogroup Q, three to haplogroup R1b, two each to haplogroups E1b1b and L, and one each to haplogroups R1a and I1. Male and female sequence-based X-STR random match probabilities were 3.28×10-7 and 1.22×10-6, respectively. The average observed and expected heterozygosities for 94 iSNPs were 0.39±0.12 and 0.39±0.13, respectively, and the combined iSNP RMP was 1.08×10-32. The combined STR and iSNP RMPs were 2.55×10-58 and 3.02×10-61 for length-based and sequence-based STR alleles, respectively. Ancestry and phenotypic SNP information, performed using the ForenSeq™ Universal Analysis Software, predicted black hair, brown eyes, and some probability of East Asian ancestry for all but one sample that clustered between European and Admixed American ancestry on a principal components analysis. These data serve as the first population assessment using the ForenSeq™ panel and highlight the value of employing sequence-based alleles for forensic DNA typing to increase heterozygosity, which is beneficial for identity testing in populations with reduced genetic diversity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Context: Lung cancer is the leading cause of cancer-related death in the U.S. A large randomized controlled trial in advanced lung cancer found a survival advantage with an early palliative care (EPC) intervention compared to standard oncologic care., Objectives: We performed retrospective analysis of our partially integrated onco-palliative care lung cancer clinic to evaluate overall survival and resource utilization., Methods: All outpatients with advanced lung cancer cared for within our institution from 2007 to 2011 were identified. Overall survival, clinical trial participation, hospice enrollment and length of stay, and chemotherapy utilization were calculated for patients treated with EPC and compared to standard oncologic care., Results: Two hundred seven patients with advanced lung cancer were identified; 82 received EPC. EPC patients had a survival advantage (11.9 vs. 10.1 months, P = 0.031), were more likely to participate in clinical trials (29% vs. 19%, P = 0.014), and median hospice length of stay was longer (38.5 vs. 24 days, P = 0.032). No difference in chemotherapy utilization or hospice enrollment was seen between the groups., Conclusion: EPC in advanced lung cancer was associated with a nearly two-month survival advantage compared to standard oncologic care. This finding provides supportive evidence to previously published reports of survival benefit with EPC intervention. Clinical trial participation rates in advanced lung cancer are lacking, and we found more frequent clinical trial participation in the early PC group. No differences were seen in chemotherapy utilization or hospice enrollment. EPC patients' longer hospice length of stay did not compromise survival., (Copyright © 2016 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

The DNA Commission of the International Society for Forensic Genetics (ISFG) is reviewing factors that need to be considered ahead of the adoption by the forensic community of short tandem repeat (STR) genotyping by massively parallel sequencing (MPS) technologies. MPS produces sequence data that provide a precise description of the repeat allele structure of a STR marker and variants that may reside in the flanking areas of the repeat region. When a STR contains a complex arrangement of repeat motifs, the level of genetic polymorphism revealed by the sequence data can increase substantially. As repeat structures can be complex and include substitutions, insertions, deletions, variable tandem repeat arrangements of multiple nucleotide motifs, and flanking region SNPs, established capillary electrophoresis (CE) allele descriptions must be supplemented by a new system of STR allele nomenclature, which retains backward compatibility with the CE data that currently populate national DNA databases and that will continue to be produced for the coming years. Thus, there is a pressing need to produce a standardized framework for describing complex sequences that enable comparison with currently used repeat allele nomenclature derived from conventional CE systems. It is important to discern three levels of information in hierarchical order (i) the sequence, (ii) the alignment, and (iii) the nomenclature of STR sequence data. We propose a sequence (text) string format the minimal requirement of data storage that laboratories should follow when adopting MPS of STRs. We further discuss the variant annotation and sequence comparison framework necessary to maintain compatibility among established and future data. This system must be easy to use and interpret by the DNA specialist, based on a universally accessible genome assembly, and in place before the uptake of MPS by the general forensic community starts to generate sequence data on a large scale. While the established nomenclature for CE-based STR analysis will remain unchanged in the future, the nomenclature of sequence-based STR genotypes will need to follow updated rules and be generated by expert systems that translate MPS sequences to match CE conventions in order to guarantee compatibility between the different generations of STR data., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)