Skeletal samples.

<p>N.R.: No remarkable change.</p

Geographical representation of excavation site.

<p>Location of <i>Hatanai</i> site in northeastern Japan (A). A map of seven cemeteries and the site where SK26 was excavated (B). A grave pit of SK26 at excavated (C) Cemetery 5 (not shown in this map) locates 50 m south of cemetery 2. All these figures were modified with permission from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012422#pone.0012422-Takigawa1" target="_blank">[23]</a>.</p

PCR detection of <i>M. leprae</i> DNA from skeletal samples.

<p>PCR analysis was performed using <i>M. leprae</i>-specific <i>hsp-70</i> and <i>16S-rRNA</i> primers for the DNA samples listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012422#pone-0012422-t001" target="_blank">Table 1</a>. PCR products were evaluated by 2% agarose gel electrophoresis. Human <i>β-globin</i> gene was also PCR amplified as a control for DNA preparation from a premolar root. NC1: a negative control for DNA purification in which DNase/RNase-free water was used as a sample for DNA extraction; NC2: a negative control for PCR in which DNase/RNase-free water was used instead of a DNA sample for PCR reaction; and PC: positive control DNA from <i>Thai 53</i> strain of <i>M. leprae</i>.</p

PCR primer sequences.

<p>Numbers in SNP nested primer indicate the coordinate location of the SNP. Numbers in non-coding regions indicate coordinate position of the primer within <i>M. leprae</i> genome (<a href="http://genolist.pasteur.fr/Leproma/" target="_blank">http://genolist.pasteur.fr/Leproma/</a>).</p

Sequence analysis of the three reported SNPs of <i>M. leprae</i> DNA.

<p>Each locus was PCR amplified and sequenced as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012422#s2" target="_blank">Materials and Methods</a>.</p

Comparison of conventional PCR and WGA-PCR.

<p>PCR was performed using a DNA sample derived from the present study (sample No. 1 shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012422#pone-0012422-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012422#pone-0012422-g003" target="_blank">Fig. 3</a>). The DNA before and after WGA (“−” and “+”, respectively) was amplified using specific nested primers and direct primers targeted for the genes, pseudogenes and non-coding regions of <i>M. leprae</i> genome as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012422#pone-0012422-t002" target="_blank">Table 2</a>. Names of the genes and pseudogenes are indicated and the coordinate positions of non-coding regions in the genome (<a href="http://genolist.pasteur.fr/Leproma/" target="_blank">http://genolist.pasteur.fr/Leproma/</a>) are indicated numerically. PC: positive control DNA from <i>Thai 53</i> strain of <i>M. leprae</i>; and NC: negative control using DNase/RNase-free water instead of a DNA sample for PCR reaction.</p