G+C content variation in selected <i>Treponema</i> strains.

<p>TPA Mexico A, TPA SS14, TPE Samoa D and <i>Treponema paraluiscuniculi</i> strain Cuniculi A were analyzed. Colored vertical lines represent 501 bp-long windows with G+C content above the 63% or below the 41% threshold. Black vertical lines represent genome locations of <i>tpr</i> genes. Stars denote 5 kb-long DNA regions with 40 or more nucleotide positions differentiating TPA and TPE strains <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001832#pntd.0001832-Cejkova1" target="_blank">[7]</a>. Please note that there is no clear association of DNA regions with different G+C content and regions differentiating TPA and TPE strains <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001832#pntd.0001832-Cejkova1" target="_blank">[7]</a> or locations of <i>tpr</i> genes.</p

A list of changes found in the TPAMA_0326 locus.

<p>Three TPA (Nichols, SS14, and Chicago) and three TPE (CDC-2, Gauthier, and Samoa D) strains were analyzed. The numbers in square brackets correspond to amino acid position in the TPAMA_0326 protein. The amino acid sequence of the encoded protein is shown in brackets.</p>1)<p>Domain prediction according to Desrosiers <i>et al.</i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001832#pntd.0001832-Desrosiers1" target="_blank">[41]</a>.</p>2)<p>G (R) was found in TPE CDC-2 strain, G (E) in TPE Gauthier, while T (N) was found in TPE Samoa D strain.</p>3)<p>15 bp (GTAGAACCACCAGCT) encode SRTTS in TPA Nichols and Chicago strain and 15 bp (GTAGCACCACCAGCT) encode SSTTS in TPA SS14 strain.</p

Two possible molecular mechanisms resulting in formation of the mosaic structure of the TPAMA_0326 locus.

<p>Green lines are nucleotide sequences specific for TPA strains (Nichols, SS14, Chicago), red ones for TPE strains (CDC-2, Gauthier, Samoa D). Vertical black lines denote corresponding positions of the TPAMA_0326 loci. A) Homologous recombination. 1. Homologous recombination between TPA and TPE loci with suggested possible recombination sites (x); arrow indicates direction of branch migration. 2. The resulting heteroduplex contains both TPA and TPE sequences; green loop denotes a 15 bp insertion present in all investigated TPA strains. 3. Recognition of mismatched DNA by MutS-MutL complex and subsequent cleavage of DNA strands by endonucleases (marked by arrows). 4. Unwinding of cleaved strands by DNA helicase (open elipse) and DNA polymerase-mediated (grey) gap filling. 5. Reparation of DNA breaks with DNA ligase. Alternatively, there is a possibility of an active DNA uptake across the cell membrane. Although no gene orthologs involved in natural competence have been identified in the TPA genomes, one cannot exclude this activity in one or several genes with unknown function. B) Gene conversion. Successive half crossing-over model. 1. First half crossing-over with suggested recombination site (x). 2. Successive second half crossing-over and the second recombination site (x). 3. Duplex recovery and degradation of both displaced ends. Please note that model B) requires the presence of two TPA-like nucleotide sequences in the donor TPE sequence.</p

A schematic representation of the TPAMA_0326 and TPAMA_0488 loci.

<p>The Mexico A sequence was compared to three TPA (Nichols, SS14, and Chicago) and three TPE CDC-2, Gauthier, Samoa D) strains. Black vertical lines represent nucleotide positions differentiating all TPA and TPE strains. Green and red vertical lines represent nucleotide changes present in all analyzed TPA and TPE strains where the TPA Mexico A sequence is identical to TPA and TPE strains, respectively. Two nucleotide positions (584, 1655) in the TP0488 locus were found only in the Nichols and Chicago strains and not in the SS14 genome (open vertical lines). The symbol Δ stands for 15-bp deletion present in the TPE and Mexico A strains in the TP0326 gene.</p

Validation of PBJelly

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047768#s2" target="_blank"><b>Results</b></a><b>.</b> Using Sanger sequencing of Dpse we validated 7 negative gap closures (A) and 45 closed gaps (B). We also compared PBJelly's gap closing sequence with the original Dmel reference (C).</p

Gap filling Improvements and categories produced by PBJelly.

<p>Histograms showing gap-size distribution in the original and upgraded (A) D .mel, (B) Dpse, (C) Mund, and (D) Caty references as well as a summary of the upgrade categories for gaps.</p

Description of sequencing data sets used.

<p>Histograms of read lengths in (A) Dmel, (B) Dpse, (C) Mund, (D) Caty. Panel (E) contains detailed metrics of each dataset.</p

A schematic of PBJelly's workflow and decision-making.

<p>(A) A flow chart of PBJelly's steps. (B) A schematic describing two hypothetical gaps supported by reads and the classifications used during the Support step. (C) A detailed flow chart for local assembly of PacBio reads in a gap region used during the assembly step.</p

Distribution of amount of sequence placed in closed gaps compared to overfilled gaps.

<p>Frequency plots of the absolute value of sequence placed into gaps subtracted from the predicted gap size in closed gaps versus overfilled gaps in (A) Dpse (B) Mund (C) Caty. Data for Dmel is not shown because synthetically inserted gaps' predicted gap sizes matched the amount of sequence that should have been placed into the gaps.</p

Sanger Validation Results Per Gap.

<p>Negative gaps are marked with an asterisk.</p