Targeted enrichment of whole-genome SNPs from highly burned skeletal remains.

Genetic assessment of highly incinerated and/or degraded human skeletal material is a persistent challenge in forensic DNA analysis, including identifying victims of mass disasters. Few studies have investigated the impact of thermal degradation on whole-genome single-nucleotide polymorphism (SNP) quality and quantity using next-generation sequencing (NGS). We present whole-genome SNP data obtained from the bones and teeth of 27 fire victims using two DNA extraction techniques. Extracts were converted to double-stranded DNA libraries then enriched for whole-genome SNPs using unpublished biotinylated RNA baits and sequenced on an Illumina NextSeq 550 platform. Raw reads were processed using the EAGER (Efficient Ancient Genome Reconstruction) pipeline, and the SNPs filtered and called using FreeBayes and GATK (v. 3.8). Mixed-effects modeling of the data suggest that SNP variability and preservation is predominantly determined by skeletal element and burn category, and not by extraction type. Whole-genome SNP data suggest that selecting long bones, hand and foot bones, and teeth subjected to temperatures <350°C are the most likely sources for higher genomic DNA yields. Furthermore, we observed an inverse correlation between the number of captured SNPs and the extent to which samples were burned, as well as a significant decrease in the total number of SNPs measured for samples subjected to temperatures >350°C. Our data complement previous analyses of burned human remains that compare extraction methods for downstream forensic applications and support the idea of adopting a modified Dabney extraction technique when traditional forensic methods fail to produce DNA yields sufficient for genetic identification.

Amplifying PCR productivity and environmental sustainability through shortened cycling protocols.

Since its inception in the 1980s, advancements in PCR technology using improved thermal cyclers, engineered DNA polymerases and commercial master mixes, have led to increased PCR productivity. Despite these advancements, PCR cycling protocols have largely remained unchanged over the same period. This study aimed to systemically evaluate the effect of reduced PCR cycling parameters on amplicon production. The 1466bp fragment from the 16S rRNA gene present in low-, medium- and high-CG bacteria was amplified using three commercially available PCR master mixes. The shortest cycling parameters required to successfully amplify the 16S fragment from all bacteria and master mixes comprised 30-cycles of 5 s denaturation, 25 s annealing, and 25 s extension. While all produced an amplicon with sufficient yield to enable downstream sequence analysis, the PCRBIO Ultra Mix in conjunction with the shortened parameters was found to achieve the highest amplicon yield across low-, medium- and high CG bacteria. Comparing the run times to that of a typical 16S PCR protocol, the shortened cycling parameters reduced the program duration by 46 % and consumed 50 % less electricity, translating into increased productivity and helping to improve laboratory environmental sustainability.

Personalized Cancer Monitoring Assay for the Detection of ctDNA in Patients with Solid Tumors.

BACKGROUND: Highly sensitive molecular assays have been developed to detect plasma-based circulating tumor DNA (ctDNA), and emerging evidence suggests their clinical utility for monitoring minimal residual disease and recurrent disease, providing prognostic information, and monitoring therapy responses in patients with solid tumors. The Invitae Personalized Cancer Monitoring™ assay uses a patient-specific, tumor-informed variant signature identified through whole exome sequencing to detect ctDNA in peripheral blood of patients with solid tumors. METHODS: The assay's tumor whole exome sequencing and ctDNA detection components were analytically validated using 250 unique human specimens and nine commercial reference samples that generated 1349 whole exome sequencing and cell-free DNA (cfDNA)-derived libraries. A comparison of tumor and germline whole exome sequencing was used to identify patient-specific tumor variant signatures and generate patient-specific panels, followed by targeted next-generation sequencing of plasma-derived cfDNA using the patient-specific panels with anchored multiplex polymerase chain reaction chemistry leveraging unique molecular identifiers. RESULTS: Whole exome sequencing resulted in overall sensitivity of 99.8% and specificity of > 99.9%. Patient-specific panels were successfully designed for all 63 samples (100%) with ≥ 20% tumor content and 24 (80%) of 30 samples with ≥ 10% tumor content. Limit of blank studies using 30 histologically normal, formalin-fixed paraffin-embedded specimens resulted in 100% expected panel design failure. The ctDNA detection component demonstrated specificity of > 99.9% and sensitivity of 96.3% for a combination of 10 ng of cfDNA input, 0.008% allele frequency, 50 variants on the patient-specific panels, and a baseline threshold. Limit of detection ranged from 0.008% allele frequency when utilizing 60 ng of cfDNA input with 18-50 variants in the patient-specific panels (> 99.9% sensitivity) with a baseline threshold, to 0.05% allele frequency when using 10 ng of cfDNA input with an 18-variant panel with a monitoring threshold (> 99.9% sensitivity). CONCLUSIONS: The Invitae Personalized Cancer Monitoring assay, featuring a flexible patient-specific panel design with 18-50 variants, demonstrated high sensitivity and specificity for detecting ctDNA at variant allele frequencies as low as 0.008%. This assay may support patient prognostic stratification, provide real-time data on therapy responses, and enable early detection of residual/recurrent disease.

Ancient mitochondrial genome diversity in South America: Contributions from Quebrada del Toro, Northwestern Argentina.

OBJECTIVES: The objective of this study was to enhance our understanding of the population history in South America, specifically Northwestern Argentina, by analyzing complete ancient mitogenomes of individuals from the Ojo de Agua archeological site (970 BP) in Quebrada del Toro (Salta, Argentina). MATERIALS AND METHODS: We analyzed teeth from four individuals from the site Ojo de Agua (970 ± 60 BP), located in Quebrada del Toro (Andean region of Northwestern Argentina). DNA extracts were converted to double-stranded DNA libraries and indexed using unique dual-indexing primer combinations. DNA libraries were then enriched for the complete mitochondrial genome, pooled at equimolar concentrations, and sequenced on an Illumina® MiSeq™ platform. Reads from high quality libraries were trimmed, merged, and then mapped to the revised Cambridge Reference Sequence. The aDNA damage patterns were assessed and contamination estimated. Finally, variants were called, filtered, and the consensus mitogenome was constructed and used for haplogroup assignment. We also compiled available mitogenome sequences from ancient and present-day populations from the Southcentral Andes and other surrounding regions in Argentina. Maximum Likelihood and Bayesian phylogenetic reconstructions were obtained using the generated dataset. RESULTS: We successfully obtained the complete mitogenome sequence from one individual with an average depth coverage of 102X. We discovered a novel haplotype that was assigned to haplogroup D1. Phylogenetic reconstructions suggests that this haplotype falls within the sister branches of the D1j lineage, forming a well-supported clade. The estimate TMRCA of this clade that includes D1j and its sister branches ranged between 12,535 and 18,669 ya. DISCUSSION: The sequence analyzed in this study represents the first ancient mitogenome from within the valley region in Northwestern Argentina. We found that a representative of a lineage highly associated with D1j was already present approximately 1000 BP in the region. Our results agree with the proposed origin of D1j in other regions north of Patagonia and independent of the Pacific coast fast migratory route, contrary to what was originally hypothesized. This study highlights the lack of information regarding pre-Hispanic genetic diversity and contributes to the knowledge about the peopling process in South America.

OBJETIVOS: El objetivo de este estudio fue contribuir a mejorar la comprensión del poblamiento de Sudamérica, específicamente del Noroeste Argentino, mediante el análisis de mitogenomas antiguos completos de individuos del sitio arqueológico Ojo de Agua (970 AP), Quebrada del Toro (Salta, Argentina). MATERIALES Y MÉTODOS: Se analizaron dientes de cuatro individuos del sitio Ojo de Agua (970 ± 60 AP), ubicado en la Quebrada del Toro (región andina del Noroeste Argentino). A partir de los extractos de ADN se armaron librerías doble cadena y se indexaron utilizando combinaciones únicas de pares de cebadores. Las librerías fueron luego enriquecidas en ADN mitocondrial, llevadas a concentraciones equimolares y secuenciadas en una plataforma Illumina® MiSeq™. Las lecturas de las librerías de alta calidad fueron recortadas, fusionadas y, posteriormente, alineadas con la Secuencia de Referencia de Cambridge revisada. Se evaluaron los patrones de daño del ADNa y se estimó la contaminación. Por último, se identificaron las variantes, se filtraron y se construyó el mitogenoma consenso, que se utilizó para la asignación de haplogrupos. Además, se recopilaron secuencias de mitogenomas disponibles para poblaciones pre-hispánicas y actuales de los Andes Centrosur y otras regiones adyacentes de Argentina. Utilizando el conjunto de datos generado, se obtuvieron reconstrucciones filogenéticas mediante Máxima Verosimilitud y estimación Bayesiana. RESULTADOS: Se obtuvo la secuencia completa del mitogenoma de un individuo con una profundidad media de 102X. Esta secuencia corresponde a un nuevo haplotipo que fue asignado al haplogrupo D1. Las reconstrucciones filogenéticas sugirieron que este haplotipo se encuentra dentro del grupo hermano del linaje D1j, conformando un clado bien soportado. La datación estimada para este clado que contiene a D1j y a todo su grupo hermano fue de entre 12.535 y 18.669 años atrás. DISCUSIÓN: La secuencia analizada en este estudio representa el primer mitogenoma antiguo de la región valliserrana del Noroeste Argentino. Se halló que un representante de un linaje altamente asociado con D1j ya estaba presente hace aproximadamente 1000 años AP en la región. Nuestros resultados concuerdan con un origen de D1j en otras regiones al norte de la Patagonia, e independientemente de la ruta migratoria rápida por la costa del Pacífico, en contraposición a lo planteado inicialmente. Este estudio pone en evidencia la falta de información que se tiene actualmente sobre la diversidad genética en tiempos prehispánicos y contribuye al conocimiento del proceso de poblamiento de Sudamérica.

PROMETHEUS: Long-Term Exacerbation and Mortality Benefits of Implementing Single-Inhaler Triple Therapy in the US COPD Population.

Background: The US population includes 24 million to 29 million people with diagnosed and undiagnosed chronic obstructive pulmonary disease (COPD). Studies have demonstrated the safety and efficacy of single-inhaler triple therapy (SITT) in reducing COPD exacerbations. Long-term population implications of SITT use have not been quantified. Objectives: This simulation-based projection aimed to estimate the potential impact of widespread SITT use on the US COPD population. Methods: Exacerbation and all-cause mortality reductions reported in the Efficacy and Safety of Triple Therapy in Obstructive Lung Disease trial (ETHOS; NCT02465567) were used to project clinical outcomes in US patients meeting ETHOS trial eligibility criteria (ETHOS-Eligible) and patients meeting a practical definition of SITT eligibility (Expanded ETHOS-Eligible). The US COPD population was modeled with 1000 simulations of patient progression over 10 years. Agent characteristics were based on literature and claims analysis of the 2016-2018 Medicare 100% fee-for-service and IBM MarketScan® databases. Agent annual characteristics reflected incident cases, changes in COPD severity, treatment, mortality, and exacerbations under status quo treatment patterns and scenarios for the adoption of SITT. The scenarios assumed the reduced exacerbation and mortality rates associated with SITT according to ETHOS trial outcomes mean values. Results: Higher than current SITT adoption over 10 years would be expected to substantially reduce COPD exacerbation-associated hospitalizations by 2 million. Applying mean improvements reported in ETHOS for SITT would extend average patient life expectancy 2.2 years for ETHOS-Eligible patients and 1.7 years for Expanded ETHOS-Eligible patients. The number needed to treat to extend the average patient life by 1 year was 8 for the ETHOS-Eligible population and 10 for the Expanded ETHOS-Eligible population. Discussion: Widespread SITT adoption may be impeded by competitive pressures from generic treatments and nonadherence, and efficacy observed in clinical trials may not occur in real-world populations. Conclusions: Assuming ETHOS treatment effects and adherence translate to clinical practice, higher than current use of SITT can substantially reduce COPD exacerbations and hospitalizations and extend survival. These results should be viewed cautiously, because the improved outcomes for SITT in the ETHOS final retrieved vital statistics data were not statistically significant for all comparator therapy groups.

Learning by doing and creation of the shared discovery curriculum.

BACKGROUND: The Michigan State College of Human Medicine began as an experiment to teach medical students in community-based settings and to create a primary care workforce for the state. Decades later, CHM faced internal and external challenges that spurred creation of a new curriculum - the Share Discovery Curriculum - founded on learning by doing and other learning theories. METHODS: A curricular design group (CDG) developed guiding principles for reform. Based on this, pedagogies and structures were selected to achieve this vision and developed into a curricular structure. Components of the first-year curriculum were piloted with a group of students and faculty members. RESULTS: Six guiding principles were endorsed, grounded in learning theories such as Dewey's Learning by Doing. Based upon these, several key features of the new curriculum emerged: learning communities; one-on-one coaches for students; symptom-based presentations for content; simulation, authentic clinical tasks, flipped classrooms, and modified practice-based learning as primary teaching modalities; early, integrated clinical and scientific learning; milestones as course learning objectives; and a multidimensional, competency-based assessment system. DISCUSSION: The process and outcomes described here are intended as an exemplar for schools undertaking curricular change. Early stakeholder engagement, faculty development, sustainable administrative systems, and managing complexity are core to the success of such endeavors.

Characterization of the immunoglobulin lambda chain locus from diverse populations reveals extensive genetic variation.

Immunoglobulins (IGs), crucial components of the adaptive immune system, are encoded by three genomic loci. However, the complexity of the IG loci severely limits the effective use of short read sequencing, limiting our knowledge of population diversity in these loci. We leveraged existing long read whole-genome sequencing (WGS) data, fosmid technology, and IG targeted single-molecule, real-time (SMRT) long-read sequencing (IG-Cap) to create haplotype-resolved assemblies of the IG Lambda (IGL) locus from 6 ethnically diverse individuals. In addition, we generated 10 diploid assemblies of IGL from a diverse cohort of individuals utilizing IG-Cap. From these 16 individuals, we identified significant allelic diversity, including 36 novel IGLV alleles. In addition, we observed highly elevated single nucleotide variation (SNV) in IGLV genes relative to IGL intergenic and genomic background SNV density. By comparing SNV calls between our high quality assemblies and existing short read datasets from the same individuals, we show a high propensity for false-positives in the short read datasets. Finally, for the first time, we nucleotide-resolved common 5-10 Kb duplications in the IGLC region that contain functional IGLJ and IGLC genes. Together these data represent a significant advancement in our understanding of genetic variation and population diversity in the IGL locus.

Student Perceptions of Effective Simulation Instructor Teaching.

SUMMARY STATEMENT: Twenty-three focus groups were held with 183 first-year medical students to assess student perceptions of effective simulation instructors during preclinical training in a medical school. Qualitative descriptive analysis guided the interpretation of focus group data. Students identified 6 areas of knowledge (schedule, student learning goals, session scenario, tasks and checklists, technique, and session purpose); 5 effective instructor skill categories (setting up the learning environment, teaching at the appropriate level, teaching technique, providing deeper context, and giving effective feedback); and 8 positive instructor attributes (enthusiasm, engaged, prepared, knowledgeable, patient, relational, transparent, and calm) instructors should have. Each category of instructor attributes, skills, and knowledge was also described in detail providing illustrative examples of what effective instruction would look like in practice from the students' perspective. Recommendations for instructor faculty development methods and topics/goals are given.

Core Entrustable Professional Activities (EPAs) and the Transition from Medical School to Residency: the Postgraduate Year One Resident Perspective.

INTRODUCTION: The Association of American Medical Colleges (AAMC) proposed thirteen core Entrustable Professional Activities (EPAs) that all graduates should be able to perform under indirect supervision upon entering residency. As an underlying premise is that graduates ready to do so will be better prepared to transition to the responsibilities of residency, we explored the relationship between postgraduate year (PGY)-1 residents' self-assessed preparedness to perform core EPAs under indirect supervision at the start of residency with their ease of transition to residency. METHODS: Using response data to a questionnaire administered in September 2019 to PGY-1 residents who graduated from AAMC core EPA pilot schools, we examined between-group differences and independent associations for each of PGY-1 position type, specialty, and "EPA-preparedness" score (proportion of EPAs the resident reported as prepared to perform under indirect supervision at the start of residency) and ease of transition to residency (from 1 = much harder to 5 = much easier than expected). RESULTS: Of 274 questionnaire respondents (19% of 1438 graduates), 241 (88% of 274) had entered PGY-1 training and completed all questionnaire items of interest. EPA-preparedness score (mean 0.71 [standard deviation 0.26]) correlated with ease of transition (3.1 [0.9]; correlation = .291, p < .001). In linear regression controlling for specialty (among other variables), EPA-preparedness score (ß-coefficient 1.08; 95% confidence interval .64-1.52; p < .001) predicted ease of transition to residency. CONCLUSION: Graduates who felt prepared to perform many of the core EPAs under indirect supervision at the start of PGY-1 training reported an easier-than-expected transition to residency. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40670-021-01370-3.

Dataset of oxygen, carbon, and strontium isotope values from the Imperial Roman site of Velia (ca. 1st-2nd c. CE), Italy.

The oxygen (δ18Ocarbonate), strontium (87Sr/86Sr), and previously unpublished carbon (δ13Ccarbonate) isotope data presented herein from the Imperial Roman site of Velia (ca. 1st to 2nd c. CE) were obtained from the dental enamel of human permanent second molars (M2). In total, the permanent M2s of 20 individuals (10 male and 10 female) were sampled at the Museo delle Civiltà in Rome (formerly the Museo Nazionale Preistorico Etnografico "L. Pigorini") and were subsequently processed and analysed at McMaster University. A subsample of teeth (n=5) was initially subjected to Fourier transform infrared spectroscopy (FTIR) analysis to assess for diagenetic alteration through calculation of crystallinity index (CI) values. Subsequently, tooth enamel was analysed for δ13Ccarbonate and δ18Ocarbonate (VPDB) using a VG OPTIMA Isocarb isotope ratio mass spectrometer (IRMS) at McMaster Research for Stable Isotopologues (MRSI), and 87Sr/86Sr was measured by dynamic multi-collection using a thermal ionization mass spectrometer (TIMS) in the School of Geography and Earth Sciences. The dental enamel isotope data presented represent the first δ18O, δ13Ccarbonate, and 87Sr/86Sr values analysed from Imperial Roman Campania to date, providing data of use for comparative analyses of δ18O, δ13C, and 87Sr/86Sr values within the region and for assisting in documenting human mobility in archaeological contexts. Full interpretation of the δ18O and 87Sr/86Sr data presented here is provided in "Imperial Roman mobility and migration at Velia (1st to 2nd c. CE) in southern Italy" [1].

Revisiting the Core Entrustable Professional Activities for Entering Residency.

The Core EPAs for Entering Residency Pilot project aimed to test the feasibility of implementing 13 entrustable professional activities (EPAs) at 10 U.S. medical schools and to gauge whether the use of the Core EPAs could improve graduates' performance early in residency. In this manuscript, the authors (members of the pilot institutions and Association of American Medical Colleges staff supporting the project evaluation) describe the schools' capacity to collect multimodal evidence about their students' performance in each of the Core EPAs and the ability of faculty committees to use those data to make decisions regarding learners' readiness for entrustment. In reviewing data for each of the Core EPAs, the authors reflected on how each activity performed as an EPA informed by how well it could be assessed and entrusted. For EPAs that did not perform well, the authors examined whether there are underlying practical and/or theoretical issues limiting its utility as a measure of student performance in medical school.

A Novel Framework for Characterizing Genomic Haplotype Diversity in the Human Immunoglobulin Heavy Chain Locus.

An incomplete ascertainment of genetic variation within the highly polymorphic immunoglobulin heavy chain locus (IGH) has hindered our ability to define genetic factors that influence antibody-mediated processes. Due to locus complexity, standard high-throughput approaches have failed to accurately and comprehensively capture IGH polymorphism. As a result, the locus has only been fully characterized two times, severely limiting our knowledge of human IGH diversity. Here, we combine targeted long-read sequencing with a novel bioinformatics tool, IGenotyper, to fully characterize IGH variation in a haplotype-specific manner. We apply this approach to eight human samples, including a haploid cell line and two mother-father-child trios, and demonstrate the ability to generate high-quality assemblies (>98% complete and >99% accurate), genotypes, and gene annotations, identifying 2 novel structural variants and 15 novel IGH alleles. We show multiplexing allows for scaling of the approach without impacting data quality, and that our genotype call sets are more accurate than short-read (>35% increase in true positives and >97% decrease in false-positives) and array/imputation-based datasets. This framework establishes a desperately needed foundation for leveraging IG genomic data to study population-level variation in antibody-mediated immunity, critical for bettering our understanding of disease risk, and responses to vaccines and therapeutics.

Assessing the growth in clinical skills using a progress clinical skills examination.

BACKGROUND: This study evaluates the generalizability of an eight-station progress clinical skills examination and assesses the growth in performance for six clinical skills domains among first- and second-year medical students over four time points during the academic year. METHODS: We conducted a generalizability study for longitudinal and cross-sectional comparisons and assessed growth in six clinical skill domains via repeated measures ANOVA over the first and second year of medical school. RESULTS: The generalizability of the examination domain scores was low but consistent with previous studies of data gathering and communication skills. Variations in case difficulty across administrations of the examination made it difficult to assess longitudinal growth. It was possible to compare students at different training levels and the interaction of training level and growth. Second-year students outperformed first-year students, but first-year students' clinical skills performance grew faster than second-year students narrowing the gap in clinical skills over the students' first year of medical school. CONCLUSIONS: Case specificity limits the ability to assess longitudinal growth in clinical skills through progress testing. Providing students with early clinical skills training and authentic clinical experiences appears to result in the rapid growth of clinical skills during the first year of medical school.

Simulation 2.0: Integrating Basic Scientists and Clinicians in a Simulation Environment.

An increasing number of medical schools are implementing curricular changes that better integrate clinical and basic sciences throughout all four years of medical school. One of the most frequently cited reasons is to improve medical student clinical reasoning skills while simultaneously aiming to decrease the attrition of basic science knowledge. Multiple pedagogical strategies have been explored to achieve this goal. We have found that simulation is a viable medium to integrate basic science within standardized patient encounters for early medical students.

Rapid detection of extra-intestinal pathogenic Escherichia coli multi-locus sequence type 127 using a specific PCR assay.

PURPOSE: Members of the ST127 uropathogenic E. coli (UPEC) clone have a high virulence potential and are also highly virulent in insect infection models. However, strains of this lineage are reported in relatively low numbers in many studies. ST127 strains are also usually widely susceptible to antibiotics and, consequently, their true prevalence may be under-recognized as they will be eradicated during empirical therapy. A genuine concern is the possibility that members of this highly virulent lineage will acquire resistance, leading to a more serious threat. The aim of this study was to design and validate a PCR assay specific to ST127. METHODOLOGY: Genomic sequences obtained from various UPEC isolates from the leading clones were used in comparative genomic analyses to allow identification of highly discriminatory sequences specific to E. coli ST127. The fliC (flagellin) and a homologue of the upaG (autotransporter adhesin) gene were identified as meeting our criteria and were used to develop a multiplex PCR assay. A total of 143 UPEC isolates representing 99 different MLST clones from three locations (North West and South West England and Riyadh, Saudi Arabia) were used to validate the PCR assay. RESULTS: The multiplex PCR readily identified all 29 E. coli ST127 isolates but, equally importantly, produced no false positives with representatives of any of the other 98 STs tested. CONCLUSION: We report the design and validation of a specific multiplex PCR for the rapid and reliable identification of ST127, which can be used for enhanced surveillance for this high-risk clone.

Mapping the origins of Imperial Roman workers (1st-4th century CE) at Vagnari, Southern Italy, using 87 Sr/86 Sr and δ18 O variability.

OBJECTIVES: We obtained the oxygen and strontium isotope composition of teeth from Roman period (1st to 4th century CE) inhabitants buried in the Vagnari cemetery (Southern Italy), and present the first strontium isotope variation map of the Italian peninsula using previously published data sets and new strontium data. We test the hypothesis that the Vagnari population was predominantly composed of local individuals, instead of migrants originating from abroad. MATERIALS AND METHODS: We analyzed the oxygen (18 O/16 O) and strontium (87 Sr/86 Sr) isotope composition of 43 teeth. We also report the 87 Sr/86 Sr composition of an additional 13 molars, 87 Sr/86 Sr values from fauna (n = 10), and soil (n = 5) samples local to the area around Vagnari. The 87 Sr/86 Sr variation map of Italy uses 87 Sr/86 Sr values obtained from previously published data sources from across Italy (n = 199). RESULTS: Converted tooth carbonate (δ18 ODW ) and 87 Sr/86 Sr data indicate that the majority of individuals buried at Vagnari were local to the region. ArcGIS bounded Inverse Distance Weighting (IDW) interpolation of the pan-Italian 87 Sr/86 Sr data set approximates the expected 87 Sr/86 Sr range of Italy's geological substratum, producing the first strontium map of the Italian peninsula. DISCUSSION: Results suggest that only 7% of individuals buried at Vagnari were born elsewhere and migrated to Vagnari, while the remaining individuals were either local to Vagnari (58%), or from the southern Italian peninsula (34%). Our results are consistent with the suggestion that Roman Imperial lower-class populations in southern Italy sustained their numbers through local reproduction measures, and not through large-scale immigration from outside the Italian peninsula.