Comparison of heterogeneous positions identified in TPA SS14 strain by Matějková et al. [14] and by the automated pipeline used in this study.

<p>^aadditional intrastrain heterogeneous genome positions identified by Matějková et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref014" target="_blank">14</a>] including 135141, 135144, 135149, 135220, 135227, 671982, 672004, 672016, 672025, 672026, 672027, 672028, 672036, 672039, 672040, 672041, 672042, 672043, 672044, 672154, 673088, 673119, 673511, 673545, 673550, and 673554 (according to the CP000805.1) were located in paralogous regions and therefore were excluded from the automated pipeline (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.s002" target="_blank">S2 Table</a>)</p><p>^bnumbers in parentheses show numbers of sequenced clones [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref014" target="_blank">14</a>] or nucleotide frequency within individual Illumina sequence reads (this study); NA—not available</p><p>^cnot present in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.t002" target="_blank">Table 2</a>; heterogeneous positions were detected in raw Illumina sequencing reads but were excluded due to study criteria</p><p>^d these heterogeneous sites were not found among Illumina reads, but were identified among 454 reads (SRX000109)</p><p>^esee also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.t003" target="_blank">Table 3</a>; independent DNA preparations showed clear differences in proportions of alternative alleles, ranging from 0.1 to 0.7</p><p>Comparison of heterogeneous positions identified in TPA SS14 strain by Matějková et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref014" target="_blank">14</a>] and by the automated pipeline used in this study.</p

Summary of the intrastrain variable sites identified within Illumina sequencing reads in investigated treponemal genomes.

<p>Illumina-identified intrastrain variable sites were verified using 454 or Sanger sequencing.</p><p>^ano intrastrain heterogeneous site were identified in the TPA Mexico A, TPE CDC-2, TPE Gauthier, TPE Fribourg-Blanc and TPLC Cuniculi A genomes</p><p>^bnonconservative amino acid replacements are not listed</p><p>^cif not indicated, localization was predicted by PSORTb</p><p>^dnot applicable</p><p>^e[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref020" target="_blank">20</a>],[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004110#pntd.0004110.ref023" target="_blank">23</a>]</p><p>^fvariable number of direct repeat (TCCTCCCCC)</p><p>Summary of the intrastrain variable sites identified within Illumina sequencing reads in investigated treponemal genomes.</p

Data analysis workflow.

<p><b>(A)</b> An automated identification pipeline and optimization process. <b>(B)</b> An application of further restrictions and verification of identified putative candidates.</p

Gene target selection for loop-mediated isothermal amplification for rapid discrimination of <i>Treponema pallidum</i> subspecies

<div><p>We show proof of concept for gene targets (<i>polA</i>, <i>tprL</i>, and TP_0619) that can be used in loop-mediated isothermal amplification (LAMP) assays to rapidly differentiate infection with any of the three <i>Treponema pallidum</i> subspecies (<i>pallidum</i> (<i>TPA</i>), <i>pertenue</i> (<i>TPE</i>), and <i>endemicum</i> (<i>TEN</i>)) and which are known to infect humans and nonhuman primates (NHPs). Four <i>TPA</i>, six human, and two NHP <i>TPE</i> strains, as well as two human <i>TEN</i> strains were used to establish and validate the LAMP assays. All three LAMP assays were highly specific for the target DNA. Amplification was rapid (5–15 min) and within a range of 10E+6 to 10E+2 of target DNA molecules. Performance in NHP clinical samples was similar to the one seen in human <i>TPE</i> strains. The newly designed LAMP assays provide proof of concept for a diagnostic tool that enhances yaws clinical diagnosis. It is highly specific for the target DNA and does not require expensive laboratory equipment. Test results can potentially be interpreted with the naked eye, which makes it suitable for the use in remote clinical settings.</p></div

Detection limits of LAMP assays.

<p>(A-C) <i>TP</i> LAMP assay involving (A) <i>TPA</i> strain Nichols with copy numbers 10E+5 to 10E+0, (B) <i>TPE</i> strain Gauthier tested in a range of 10E+6 to 10E+0 copies, and (C) <i>TEN</i> strain Bosnia A in 10E+6 to 10E+0 copy numbers. (D-E) <i>TPA</i> LAMP assay run with <i>TPA</i> strain Nichols from 10E+5 to 10E+0 copy numbers, and the same LAMP assay run with (E) <i>TEN</i> strain Bosnia A in copy numbers ranging from 10E+6 to 10E+0. (F-G) <i>TPE</i>/<i>TEN</i> LAMP assay involving (E) <i>TPE</i> strain Gauthier tested in 10E+6 to 10E+0 copy numbers, and (F) <i>TEN</i> strain Bosnia A tested in 10E+6 to 10E+0 copies. Red cross = Negative control, <i>TPA</i> strain Nichols (blue), <i>TPE</i> strain Gauthier (green), <i>TEN</i> strain Bosnia A (black); symbols represent copy numbers: circle with cross = 10E+6, hexagon = 10E+5, square turned = 10E+4, square = 10E+3, circle = 10E+2, down-pointing triangle = 10E+1, up-pointing triangle = 10E+0.</p

Detection limits for the three LAMP assays.

<p>Corresponding graphs can be found in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006396#pntd.0006396.g002" target="_blank">Fig 2</a>. + = exponential amplification, (+) = no exponential amplification,— = no amplification.</p

Oligonucleotide primers used in this study.

<p>Oligonucleotide primers used in this study.</p

MOESM2 of Reanalysis of Chinese Treponema pallidum samples: all Chinese samples cluster with SS14-like group of syphilis-causing treponemes

Additional file 2. Mapping statistics of input read pairs mapped to the reference genomes. Sequencing reads derived from the Chinese strain SRA data were mapped to the Treponema pallidum subsp. pallidum (TPA) SS14 and Nichols reference genomes [6] and to the rabbit genome (Statistics was calculated from post-processed mappings; repetitive and homologous sequences and PCR duplicated reads were excluded from the statistics)

Performance characteristics of the three LAMP assays.

<p>(A) LAMP targeting the <i>polA</i> gene. All <i>TP</i> strains become positive. (B) LAMP assay targeting the <i>tprL</i> locus, which results in amplification of <i>TPA</i> and <i>TEN</i> strains. (C) Only <i>TPE</i> and <i>TEN</i> strains generate positive results in the LAMP assay targeting the TP_0619 locus. Note that the strain material in the three assays had different <i>TP</i> copy numbers as indicated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006396#pntd.0006396.s004" target="_blank">S1 Table</a>. Data points are presented as mean±SEM values. Red cross = Negative control; <i>TPA</i> strains (blue) up-pointing triangle = Mexico A, down-pointing triangle = SS14, circle = Nichols, square = Seattle 81–4; <i>TPE</i> strains (green) square = Gauthier, square turned = Sei Geringing K403, hexagon = Kampung Dalan K363, circle = Samoa D, circle with cross = CDC-1, square with cross CDC-2; <i>TPE</i> simian strains (purple) cross = Fribourg-Blanc, star = RNP; <i>TEN</i> strains (black) square = Bosnia A, circle with cross = Iraq B.</p

LAMP assays will simplify the existing test algorithm for human treponematoses.

<p>The test algorithm proposed by Ratnam 2005 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006396#pntd.0006396.ref042" target="_blank">42</a>] is shown in thin lines. The new LAMP assays for diagnosis of yaws is shown in bold lines and will simplify and accelerate yaws diagnosis. Furthermore, it enables the clinician to rapidly discriminate <i>TPE</i> infection from infection with other <i>TP</i> subspecies or pathogens.</p