A mobile laboratory for ancient DNA analysis

Abstract

Mobile devices for on-field DNA analysis have been used for medical diagnostics at the point-of-care, forensic investigations and environmental surveys, but still have to be validated for ancient DNA studies. We report here on a mobile laboratory that we setup using commercially available devices, including a compact real-time PCR machine, and describe procedures to perform DNA extraction and analysis from a variety of archeological samples within 4 hours. The process is carried out on 50 mg samples that are identified at the species level using custom TaqMan real-time PCR assays for mitochondrial DNA fragments. We evaluated the potential of this approach in museums lacking facilities for DNA studies by analyzing samples from the Enlène (MIS 2 layer) and the Portel-Ouest cave (MIS 3 deposits), and also performed experiments during an excavation campaign at the Roc-en-Pail (MIS 5) open-air site. Enlène Bovinae bone samples only yielded DNA for the extinct steppe bison (Bison priscus), whereas Portel-Ouest cave coprolites contained cave hyena (Crocuta crocuta spelaea) DNA together, for some of them, with DNA for the European bison sister species/subspecies (Bison schoetensacki/Bb1-X), thus highlighting the cave hyena diet. Roc-en-Pail Bovinae bone and tooth samples also contained DNA for the Bison schoetensacki/Bb1-X clade, and Cervidae bone samples only yielded reindeer (Rangifer tarandus) DNA. Subsequent DNA sequencing analyses confirmed that correct species identification had been achieved using our TaqMan assays, hence validating these assays for future studies. We conclude that our approach enables the rapid genetic characterization of tens of millennia-old archeological samples and is expected to be useful for the on-site screening of museums and freshly excavated samples for DNA content. Because our mobile laboratory is made up of commercially available instruments, this approach is easily accessible to other investigators.

Fig 1. Information about archeological sites.

(a) Location of sites. (b) Samples studied in each site for DNA content. (c) Enlène cave. Paving stones in the Salle-du-Fond. (d) Portel-Ouest cave. The 5-m thick archeological deposit. (e) Roc-en-Pail open-air site during the 2018 excavation campaign.

Fig 2. Overview of the device and experimental workflow.

The left panel shows the heater-mixer used for sample incubation (1), the mini-centrifuge used for DNA extraction (2), and the real-time PCR instrument (3). The right panel summarizes the overall experimental procedure.

Fig 3. Validation of experimental procedures.

(a, b) Real-time PCR analysis of a steppe bison (Bison priscus) bone sample [48] incubated 1 h, 3 h or 16 h (a) and 1 h, 2 h, or 16 h (b) before being processed for DNA extraction. The green, purple, blue, and red amplification plots display results obtained using 1 μl of the steppe bison DNA extracts with the Bison priscus TaqMan assay. Negative controls: PCR blank, mock and cave hyena DNA extracts (1 μl) analyzed using the Bison priscus TaqMan assay. (c, d) Real-time PCR analysis of a cave hyena (Crocuta crocuta) coprolite [11] incubated 1 h, 3 h or 16 h (c) and 1 h, 2 h, or 16 h (d) before being processed for DNA extraction. The green, purple, blue, and red amplification plots display results obtained using 1 μl of the coprolite DNA extract with TaqMan assays for Crocuta crocuta and Cervus elaphus DNA, as indicated. Negative controls: PCR blank, mock and bison bone DNA extracts (1 μl) analyzed using the same TaqMan assays. (e, f) Electrophoregrams for the Bison priscus bone sample (e, 1-μl aliquot) and the Crocuta crocuta coprolite (f, 0.3-μl aliquot) DNA extracts obtained after a 2-h incubation period. LM: lower marker; UM; upper marker.

Fig 4. DNA analysis of a bone sample from the Enlène cave.

The figure shows real-time PCR data obtained from serial dilutions (from 0.1 to 0.9 μl) of the DNA extract using the Bison priscus and the Bison schoetensacki TaqMan assays. The distinctive amplification plots obtained with the two assays indicate that the sample corresponds to a Bison priscus bone. Negative controls: PCR blank and mock extract.

Fig 5. DNA sequence data for Enlène samples.

The upper part of the figure displays sequence logos derived from 27,058 (Enlène 6176), 19,361 (Enlène 6178), and 29,097 (Enlène 6179) DNA reads. Only the sequence located between the PCR primers is shown. At each position, the overall height of the stack indicates the sequence conservation, and the height of the letters the relative frequency of the nucleotides; the upper letter corresponds to the predominant nucleotide. The lower part of the figure shows the orthologous reference sequences of the Bison pricus (NC_027233), Bison schoetensacki (NC_033873), Bison bonasus (NC_014044), and Bos primigenius (NC_013996) mitochondrial genomes. For each Enlène sample, the consensus sequence is identical to the Bison priscus reference sequence. Dots indicate sequence identity.

Fig 6. DNA analysis of cave hyena (Crocuta crocuta) coprolites from the Portel cave.

(a) Real-time PCR data obtained with the Crocuta crocuta TaqMan assay using 2 μl of each DNA extract. Negative controls correspond to PCR blank and mock DNA extract. (b) Real-time PCR data obtained with the Bison schoetensacki TaqMan assay using 2 μl of the same DNA extracts. Negative controls: PCR blank and mock extract.

Fig 7. DNA Bovinae sequence data for Le Portel samples.

The upper part of the figure displays sequence logos derived from 26,130 (Le Portel T2) and 55,101 (Le Portel T14) DNA reads. Only the sequence located between the PCR primers is shown. At each position, the upper letter corresponds to the predominant nucleotide. The lower part of the figure shows the orthologous reference sequences of the Bison schoetensacki (NC_033873), Bison pricus (NC_027233), Bison bonasus (NC_014044), and Bos primigenius (NC_013996) mitochondrial genomes. For the each Le Portel sample, the consensus sequence is identical to the Bison schoetensacki reference sequence. Dots indicate sequence identity.

Fig 8

DNA analysis of Roc-en-Pail bone samples during an excavation campaign (a) Analysis of Roc-en-Pail 32 and 529 samples using the Bison schoetensacki TaqMan assay. (b) Analysis of Roc-en-Pail 10, 18, 260, and 398 samples using the Rangifer tarandus TaqMan assay. All assays were performed using 0.9 μl of DNA extract. Negative controls: PCR blank and mock extract.