Monitoring DNA Contamination in Handled vs. Directly Excavated Ancient Human Skeletal Remains

<div><p>Bones, teeth and hair are often the only physical evidence of human or animal presence at an archaeological site; they are also the most widely used sources of samples for ancient DNA (aDNA) analysis. Unfortunately, the DNA extracted from ancient samples, already scarce and highly degraded, is widely susceptible to exogenous contaminations that can affect the reliability of aDNA studies. We evaluated the molecular effects of sample handling on five human skeletons freshly excavated from a cemetery dated between the 11 to the 14^th century. We collected specimens from several skeletal areas (teeth, ribs, femurs and ulnas) from each individual burial. We then divided the samples into two different sets: one labeled as “virgin samples” (i.e. samples that were taken by archaeologists under contamination-controlled conditions and then immediately sent to the laboratory for genetic analyses), and the second called “lab samples”(i.e. samples that were handled without any particular precautions and subject to normal washing, handling and measuring procedures in the osteological lab). Our results show that genetic profiles from “lab samples” are incomplete or ambiguous in the different skeletal areas while a different outcome is observed in the “virgin samples” set. Generally, all specimens from different skeletal areas in the exception of teeth present incongruent results between “lab” and “virgin” samples. Therefore teeth are less prone to contamination than the other skeletal areas we analyzed and may be considered a material of choice for classical aDNA studies. In addition, we showed that bones can also be a good candidate for human aDNA analysis if they come directly from the excavation site and are accompanied by a clear taphonomic history.</p> </div

Ancient samples HVS-I haplotypes between positions 16024 and 16384 and putative haplogroup classification for each burial and each skeletal area.

<p>In each haplotype, only positions that differ from CRS are listed. CRS means no differences.</p

Results of quantitative Real-time PCR results.

<p>Number of molecules per microliter (mol/µl) of the target DNA in the extracts are listed for each individual and each skeletal area in both “virgin” and “lab” sample sets. Mean values and standard deviations (mol/µl) calculated for each “virgin” sample and the corresponding “lab” sample are reported in the last two columns.</p

HVS-I haplotypes of modern samples between positions 16024 and 16384 and putative haplogroup classification.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052524#pone-0052524-t002" target="_blank">table 2</a> for further details.</p

Maternal DNA lineages at the gate of Europe in the 10^th century AD

<div><p>Given the paucity of archaeogenetic data available for medieval European populations in comparison to other historical periods, the genetic landscape of this age appears as a puzzle of dispersed, small, known pieces. In particular, Southeastern Europe has been scarcely investigated to date. In this paper, we report the study of mitochondrial DNA in 10^th century AD human samples from Capidava necropolis, located in Dobruja (Southeastern Romania, Southeastern Europe). This geographical region is particularly interesting because of the extensive population flux following diverse migration routes, and the complex interactions between distinct population groups during the medieval period. We successfully amplified and typed the mitochondrial control region of 10 individuals. For five of them, we also reconstructed the complete mitochondrial genomes using hybridization-based DNA capture combined with Next Generation Sequencing. We have portrayed the genetic structure of the Capidava medieval population, represented by 10 individuals displaying 8 haplotypes (U5a1c2a, V1a, R0a2’3, H1, U3a, N9a9, H5e1a1, and H13a1a3). Remarkable for this site is the presence of both Central Asiatic (N9a) and common European mtDNA haplotypes, establishing Capidava as a point of convergence between East and West. The distribution of mtDNA lineages in the necropolis highlighted the existence of two groups of two individuals with close maternal relationships as they share the same haplotypes. We also sketch, using comparative statistical and population genetic analyses, the genetic relationships between the investigated dataset and other medieval and modern Eurasian populations.</p></div

MDS plot of Romanian medieval population and modern Eurasian populations.

<p>Stress value is 0.1323 and non-metric fit (R²) is 0.982, values that highlight a good fit between the two-dimensional graph and the original distance matrix. The linearized Slatkin F_ST values and population information are presented in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193578#pone.0193578.s013" target="_blank">S9 Table</a>.</p

PCA plot of the first two components (34.82% of variance) of medieval populations.

<p>The PCA based on mtDNA haplogroup frequencies of the 15 medieval populations shows a roughly clustering of the ancient populations from Romania (ROU-med) and medieval Cumanians from Hungary (HUN-Cum). The other medieval populations from Southern Europe are clustered together, as are those from the North of Europe. The abbreviations, references and haplogroup frequencies are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193578#pone.0193578.s009" target="_blank">S5 Table</a>.</p

Electronic Supplementary Information from Genome diversity in the Neolithic Globular Amphorae culture and the spread of Indo-European languages

Supplementary Materials, 16 supplementary tables and appendi

Levelplot of the linearized Slatkin population differentiation (F_ST) values and significant p values.

<p>Lower left corner: significant p values (< 0.05) are indicated in green. Upper right corner: larger Slatkin F_ST values indicating greater genetic distances are marked by dark red shades. The exact F_ST and p values and population information are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193578#pone.0193578.s010" target="_blank">S6 Table</a>.</p

PCA of the investigated medieval and present-day populations.

<p>The PCA is based on mtDNA haplogroup frequencies of the medieval population from Romania and 35 modern populations from Eurasia and shows PC1 and PC2. The haplogroup frequencies and population information are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193578#pone.0193578.s012" target="_blank">S8 Table</a>.</p