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In forensic DNA casework, a highly accurate real-time quantitative polymerase chain reaction (qPCR) assay is recommended per the Scientific Working Group on DNA Analysis Methods (SWGDAM) (SWGDAM Validation Guidelines for DNA Analysis Methods [1]) to determine whether a DNA sample is of sufficient quantity and robust quality to move forward with downstream short tandem repeats (STR) or sequencing analyses. Most of these assays rely on a standard curve, referred to herein and traditionally as absolute qPCR, in which an unknown is compared, relative to that curve. However, one fundamental issue with absolute qPCR is the quantifiable concentration of commercial assay standards can vary depending on (1) origin, i.e., whether from a cell line or a human subject, (2) supplier, (3) lot number, (4) shipping method, etc. In 2018, the National Institute for Standards and Technology (NIST) released a human DNA standard reference material for evaluating qPCR quantification standards, Standard Reference Material (SRM) 2372a, Romsos et al. (2018) [2] which contains three well-characterized human genomic DNA samples: Component A) a single male 1 donor, Component B) a single female 1 donor, and Component C) a 1:3 male 2 :female 2 donor, each with certification data for nDNA and informational mitochondrial DNA(mtDNA)/nuclear DNA (nDNA) ratio data. The SRM 2372a was used to assess four qPCR assays: (1) Quantifiler Trio (Thermo Fisher Scientific, Waltham, MA) for nDNA quantification, (2) NovaQUANT (EMD Millipore Corporation, San Diego, CA) for nDNA and mtDNA quantification, (3) a custom duplex mtDNA assay, and (4) a custom triplex mtDNA assay. Additionally, extracts from eighteen (18) skeletal remains were tested with the latter three assays for concordance of DNA concentration and with assays (2) and (3), for the degradation state. Our assessment revealed that an accurate, efficient, and reproducible qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the qPCR chemistry, and (3) the specific primers, and probes (if applicable), used in an assay. Our findings indicate qPCR assays may not always quantify as expected and that performance of each lot should be verified using a well-characterized DNA standard such as the NIST SRM 2372a and adjusted if warranted., (Published by Elsevier B.V.)

In 1932, seven burials were discovered on a Texas plantation that was originally the site of a 17th-century Caddo Indian village. Of the seven excavated graves, one set of remains (an adult male) was notably buried in a manner inconsistent with traditional Caddoan burial practices and has long been purported to be the remains of Sieur de Marle (a member of the French explorer La Salle's last expedition). Diary accounts of La Salle's expedition scribe report that Sieur de Marle died along a river near an Indian village during a trek to Canada to find help for colonists left behind at the ill-fated Fort St. Louis. Additionally, two lead projectiles recovered from the grave were ballistically analyzed and determined to be consistent with ammunition used in 17th-century weaponry. In the 1980s, anthropologists requested access to the remains for study, but the skull was missing. Cranial measurements recorded in 1940 and 1962 (by two independent anthropologists) were used to investigate the ancestry of this individual; and the Giles-Elliot (G-E) discriminant function was calculated to be 18.1, within the Anglo-European range. Dietary isotope testing on non-cranial skeletal elements determined that this unknown male's diet was rich in animal/marine protein sources, which differs appreciably from Caddo Indian populations of that time period. In order to genetically assess this individual's biogeographic ancestry and to provide further support that this individual is of European descent, mitochondrial DNA (mtDNA) sequencing was performed using the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel. mtDNA sequencing of multiple sections from two different long bones yielded compiled results consistent with either Haplogroup H or R, both predominantly European mtDNA haplogroups. Further anthropological calculations were conducted using cranial measurements, FORDISC™ software, and discriminant function analysis. Two-way, four-way, and multigroup discriminant function analyses further classify this set of unidentified remains as being White (European) in origin, with posterior probabilities of 0.999, 0.881 and 0.986, respectively. Combined with historical records of Sieur de Marle's death, as well as overlays of historical and contemporary maps which demonstrate that the plantation site aligns with Joutel's diary accounts of de Marle's burial, these collective results support that these remains are of a European male and may possibly belong to this prominent member of La Salle's expedition team., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)