Additional file 4: of INSaFLU: an automated open web-based bioinformatics suite “from-reads” for influenza whole-genome-sequencing-based surveillance

Table S2. A. Matrix of pairwise nucleotide differences between whole-genome consensus sequences obtained using INSaFLU versus IRMA for 137 A(H3N2) viruses from dataset 1. B. Matrix of pairwise nucleotide differences between whole-genome consensus sequences obtained using INSaFLU versus IRMA for 39 A(H1N1pdm09) viruses from dataset 2. (XLSX 100 kb

Additional file 2: of INSaFLU: an automated open web-based bioinformatics suite “from-reads” for influenza whole-genome-sequencing-based surveillance

Table S1. A. INSaFLU genetic markers for type and subtype/lineage identification (“influenza_typing” database). B. INSaFLU genetic markers for the assignment of segments (and references) to draft contigs (“influenza_assign_segments2contigs” database). GISAID acknowledgement tables are included in these tables. (XLSX 45 kb

Additional file 3: of INSaFLU: an automated open web-based bioinformatics suite “from-reads” for influenza whole-genome-sequencing-based surveillance

Figure S1. INSaFLU graphical output plotting the number of iSNVs at frequencies 1–50% (minor iSNVs) and 50–90% obtained for dataset 1. Figure S2. INSaFLU testing with artificial mixtures of A(H3N2) viruses. A. INSaFLU graphical output plotting the number of iSNVs at frequencies 1–50% (minor iSNVs) and 50–90%. B. Phylogenetic tree based on “whole-genome” consensus sequences obtained for dataset 3. (PDF 977 kb

Phase variation mediated by variable homopolymeric tracts.

<p>Panel A. The graph shows the evolution throughout passaging of the percentage of sequence reads with different ‘A’ counts in the homopolymeric tract upstream from CT533/<i>lpxC</i> for the strain E/CS1025/11. The poly(A) tract corresponds to poly(T) in the annotated leading strand. Panel B. Schematic view of the putative promoter region of CT533/<i>lpxC</i>. The predicted transcription start site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref126" target="_blank">126</a>] is labeled by +1. The variable poly(A) tract (in bold) falls between the predicted -35 and -10 hexamers (underlined). BLAST analyses revealed the existence of variable number of ‘A’ counts in <i>C</i>. <i>trachomatis</i> genomes, and also that the nucleotide indicated with an arrow is deleted exclusively in all LGV strains. Panel C. The graph shows the percentage of sequence reads with different ‘G’ counts in the variable homopolymeric tract of CT166 found in the initial populations of the epithelial-genital strains. “G” counts of nine correspond to an “ON” protein. Panel D. Schematic view of the four positions (numbers 1 to 4) relative to a gene at which contingency loci (e.g., homopolymeric tracts) can cause phase variation (adapted from van der Woude MW and Bäumler AJ, Clin Microbiol Rev <b>17</b>:581–611, 2004 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref152" target="_blank">152</a>]). Whereas positions 1 and 2 are associated with transcription initiation and position 4 with translation (ON/OFF), the mechanism regarding the position 3 is not completely disclosed. We found heterogeneity in length within homopolymeric tracts located in positions 2 (for CT533/<i>lpxC</i>) (blue), 3 (for CT043/<i>slc1</i> and the operon CT134-CT135), and 4 (for CT166—cytotoxin) (red).</p

Comparative analysis of global gene expression (RNA-seq) between D/CT135-positive and D/CT135-negative populations.

<p>Panels A-B. Comparison of gene expression between biological replicates for the D/CT135-positive (A) and D/CT135-negative (B) populations. Pearson correlation coefficients are shown. Panel C. Comparison of gene expression between the D/CT135-negative and D/CT135-positive populations. The red points mark genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected <i>P</i>-values were below 0.05. For panels A to C, axes are log₁₀-transformed normalized expression levels (FPKM). Panel D. Volcano plot of –log₂ fold change (D/CT135-positive <i>versus</i> D/CT135-negative) <i>versus</i> –log₁₀ adjusted <i>P</i>-values. In order to better fit the scale to data, corrected <i>P</i>-values ≤10⁻³ were set as 10⁻³. Points in red indicate genes and the non-coding RNA for which the fold change of expression exceeds two-fold and the FDR-corrected <i>P</i>-values were below 0.05.</p

Impact of <i>in vitro</i> passaging on the <i>C</i>. <i>trachomatis</i> growth kinetics.

<p>Panels A and B. Comparison of the growth rates and doubling times between ancestral (grey) and evolved populations (black). The percentage values above the bars correspond to the growth rate increment of the evolved population relatively to the ancestral. Panel C. Comparison of the one-step growth curve between D/CS637/11 CT135-positive and CT135-negative strains. Cells grown in the same conditions were infected at a MOI of 1, and cell scrapings were collected over time after infection for analysis of inclusion-forming units (IFUs). The black line represents the evolved CT135-negative D/CS637/11 strain, whereas the grey line represents the ancestor CT135-positive strain. The shaded area indicates the time points chosen for RNA-seq differential expression comparative analyses.</p

Mutational scenario throughout experimental evolution.

<p>Panel A. Chromosomal location of the genomic alterations observed during the <i>in vitro</i> passaging. The chromosomal position of each mutation (scale adjusted and given by the locus name) and the type of mutation event (inactivating events represented in red) are shown for each strain (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.s005" target="_blank">S3 Table</a> for details). Inactivating SNPs or indels refer to events leading to protein truncation (regardless the length of the resulting protein). For the strain D/CS637/11, the CT135 inactivating event involved the entire gene deletion between direct repeats (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.g003" target="_blank">Fig 3</a>). <b>Panel B</b>. Dynamics of the emergence and spread of mutations and their frequency in the evolving bacterial populations. For each time-point (passages 5–7, 10, 20, 30, 50 and 100), circular graphs show the frequency of the mutations in the bacterial population, where each color represents a different mutated locus. The number of bacterial generations was estimated taking into account the minimum and maximum values of the mean doubling time of the strains analyzed at each time-point, and assuming a conservative approach by considering 15 hours of exponential phase <i>per</i> bacterial life-cycle (i.e, <i>per</i> passage). Loci designations are based on genome annotation of the D/UW3 strain (GenBank accession number NC_000117).</p

Genes and a non-coding RNA found to be down-regulated in the serovar D CT135-negative strain.

<p>^a Loci nomenclature and numbering refer essentially to the annotated genome from the D/UW3 strain (GenBank accession number NC_000117).</p><p>^b Loci in bold are potentially down-regulated on behalf of the CT135 loss. Non-bold loci (also in parentheses) were previously found to be transcriptionally regulated by the plasmid (namely by the plasmid-encoded protein Pgp4) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref051" target="_blank">51</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref174" target="_blank">174</a>]. In the present study, the down-regulation of this latter set of genes may be associated with Pgp4 as it was found to display lower expression levels in the D/CS637/11 CT135-negative population.</p><p>^cAdjusted P-values < 0.05 and a fold-change cutoff > 2. Genes are ordered according the magnitude of differential expression.</p><p>^d Refers to the previously identified sRNA ctrR0332 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref126" target="_blank">126</a>] that actually comprehends two sRNAs (likely processed from a larger transcript). In the D/UW-3 annotation, it is located inside a non-existing but previously annotated ORF (CT081). Both sRNA were similarly down-regulated.</p><p>^e CT082 was suggested to be up-regulated in the <i>pgp4</i> knockout mutants [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref051" target="_blank">51</a>]. As in the present study it was found to be strongly down-regulated even with a slight <i>pgp4</i> expression decrease, we assumed this result as a consequence of CT135 loss.</p><p>^f These genes are likely expressed in the same transcriptional unit as other down-regulated contiguous genes [also marked].</p><p>^g Although it is believed that the CT143 gene is coordinately expressed with the flanking genes CT142 and CT144 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref114" target="_blank">114</a>], the obtained differential expression value was slightly below the cutoff.</p><p>Genes and a non-coding RNA found to be down-regulated in the serovar D CT135-negative strain.</p

Schematic representation of the CT135 deletion in the serovar D strain.

<p>The inactivating event of CT135 involved the complete gene deletion between direct repeats (in blue) and the putative formation of a fusion gene enrolling the two flanking genes (CT134 and CT136). The underlying mechanism likely relied in one of three major pathways: A—intermolecular crossing over between direct repeats followed by recombination (yielding both a tandem duplication and a deletion); B—looping out in between direct repeats followed by recombination; and C—DNA polymerase slippage during DNA replication [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref089" target="_blank">89</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref091" target="_blank">91</a>]. The figure also shows the position of all CT135 frameshift mutations (labeled by Ψ) reported here and elsewhere [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref046" target="_blank">46</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref048" target="_blank">48</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref131" target="_blank">131</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref149" target="_blank">149</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref150" target="_blank">150</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref164" target="_blank">164</a>], demonstrating that the strains evolved towards CT135 inactivation regardless the “genetic pathway” that drove that inactivation The bilobal hydrophobic domains that putatively enable the insertion of the CT134 and CT135 proteins into the inclusion membrane [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref033" target="_blank">33</a>] are highlighted in grey.</p

CT135 mRNA decay analysis.

<p>Panel A shows the comparison of the relative amount of transcripts at 4 h post-infection (pi) and after 10 min of transcriptional blockage with rifampicin (10 μg/ml) between the ancestral (grey) and the evolved populations (black). The assay was performed for all strains with emergent CT135-negative clones (i.e., all epithelial-genital isolates) and for the strain L2b/CS19/08 (control). The number of transcripts was quantified by independent RT-qPCR targeting the two genes of the operon CT134-CT135 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#sec002" target="_blank">methods</a> for details), except for serovar D strain as the evolved population lacks CT135. Data was normalized against the number of <i>C</i>. <i>trachomatis</i> genomes quantified on the corresponding DNA samples. In order to facilitate the comparative analysis, the normalized value before rifampicin treatment (4h pi) was arbitrarily set to 1. Panel B shows the mRNA half-life times calculated based on the fit of an exponential decay between the quantified values at 4h pi and the values calculated 10 minutes after the transcriptional arrest.</p