DNA quantification by Nanodrop Spectrophotometer

Analysis data of DNA yield from buccal swabs and wing punches harvested from live bats using nucleic acid quantification as well as DNA yields from wing tissue preserved in three media: ethanol, NaCl-saturated dimethyl sulfoxide (DMSO), and silica desiccant

Estimated coefficients of the effect of wing punch preservation methods on the natural logarithm of gene copies detected after accounting for differences between qPCR runs and among individual bats.

<p>Estimated coefficients of the effect of wing punch preservation methods on the natural logarithm of gene copies detected after accounting for differences between qPCR runs and among individual bats.</p

Relationship between the median number of copies of the single-copy nuclear gene obtained from repeated quantitative PCR reactions and concentration of whole DNA (wing punches in three preservation treatments).

<p>The correlation was significant whether including (<i>r</i> = 0.32, <i>p</i> = 0.018), or excluding the outlier measurements from sample individual ALR096 (<i>r</i> = 0.47, <i>p</i> = 0.00034).</p

Number of copies of single-copy nuclear gene obtained through quantitative PCR (qPCR) amplifications from different sample types and methods of preservation.

<p>Results are presented sorted by qPCR run. The models that best fit both datasets accounted for group-level effects of runs and individuals sampled (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118994#pone.0118994.t001" target="_blank">Table 1</a>). A. Comparison between sample types obtained from the same individuals (no significant effect of sample type was found, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118994#pone.0118994.t001" target="_blank">Table 1</a>). B. Comparison between preservation treatments for wing punch samples obtained from the same individuals (ethanol and dry preservation were significantly better than DMSO preservation, see Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118994#pone.0118994.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118994#pone.0118994.t002" target="_blank">2</a>).</p

DNA quantification qPCR

Analysis data of DNA yield from buccal swabs and wing punches harvested from live bats using quantitative PCR for a single-copy nuclear locus

DNA concentration measured using nanodrop spectrophotometer.

<p>A. Comparison between sample types obtained from the same individuals (<i>t</i>₍₂₂₎ = 6.55, <i>P</i> = 1.254e-06). B. Comparison between preservation treatments for wing punch samples obtained from the same individuals (individual effect <i>F</i>_{(20, 36)} = 5.250, <i>MSE</i> = 121.8, <i>P</i> = 8.33e-06; treatment preservation effect <i>F</i>_{(2, 36)} = 6.978, <i>MSE</i> = 161.9, <i>P</i> = 0.00275).</p

Models of the natural logarithm of nuclear gene copies detected through qPCR as a function of the sample type (<i>N</i>_{<i>observations</i>} = 90, <i>N</i>_{<i>individual bats</i>} = 12, buccal swab brush or wing punch.

<p>This includes individual WCL025, not shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118994#pone.0118994.s001" target="_blank">S1A Fig.</a> because it only had a wing punch), or the preservation medium for wing punches (<i>N</i>_{<i>observations</i>} = 228, <i>N</i>_{<i>individual bats</i>} = 21, silica beads, ethanol, or DMSO. This includes individual ALR074, not shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118994#pone.0118994.s001" target="_blank">S1B Fig.</a> because it only had a wing punch preserved in ethanol). No-predictor models serve as null hypotheses of no effect of the sample type or preservation medium on the number of <i>rag2</i> copies detected using qPCR. Group-level intercepts of qPCR run and/or individual bat sampled serve to capture effects of unmeasured variability from subtle differences in qPCR starting conditions and bat behavior. Significance was measured using a likelihood ratio test of the model with predictors to its null. Models with different sample types or preservation media as predictors can be compared to one another using the Akaike Information Criterion (AIC). The model with the lowest AIC is the best fit to the data. DF = model degrees of freedom, logLik = log-likelihood of model fit to the data, and LRT = likelihood ratio test statistic.</p><p>Models of the natural logarithm of nuclear gene copies detected through qPCR as a function of the sample type (<i>N</i>_{<i>observations</i>} = 90, <i>N</i>_{<i>individual bats</i>} = 12, buccal swab brush or wing punch.</p

Detecting the Immune System Response of a 500 Year-Old Inca Mummy

<div><p>Disease detection in historical samples currently relies on DNA extraction and amplification, or immunoassays. These techniques only establish pathogen presence rather than active disease. We report the first use of shotgun proteomics to detect the protein expression profile of buccal swabs and cloth samples from two 500-year-old Andean mummies. The profile of one of the mummies is consistent with immune system response to severe pulmonary bacterial infection at the time of death. Presence of a probably pathogenic <em>Mycobacterium sp</em>. in one buccal swab was confirmed by DNA amplification, sequencing, and phylogenetic analyses. Our study provides positive evidence of active pathogenic infection in an ancient sample for the first time. The protocol introduced here is less susceptible to contamination than DNA-based or immunoassay-based studies. In scarce forensic samples, shotgun proteomics narrows the range of pathogens to detect using DNA assays, reducing cost. This analytical technique can be broadly applied for detecting infection in ancient samples to answer questions on the historical ecology of specific pathogens, as well as in medico-legal cases when active pathogenic infection is suspected.</p> </div

Maximum likelihood phylogeny.

<p>Maximum likelihood phylogeny and bootstrap support values based on 1000 pseudoreplicates of the alignment of <i>hsp65</i> gene nucleotide sequences. Sequences generated from the Maiden’s swab samples are shown in bold, larger font, and marked with an asterisk.</p

Primers used for PCR amplification from DNA extracted from swab samples.

<p>Primers used for PCR amplification from DNA extracted from swab samples.</p