Blastn alignment of the re-sequenced nucleotide sequence (query) with the original genomic sequence (subject) of <i>hp1019</i>.

<p>The annotated gene <i>hp1018</i> is marked in grey. The letter ‘r’ represents (‘a’ OR ‘g’), while the letter ‘k’ represents (‘t’ OR ‘g’). The inserted guanidine is printed white on black. Numbers give residue positions. The amino acid translation is given in single letter code for Hp1018, starting at position 1081440, and for Hp1019, starting at position 1081537. The predicted most likely signal peptidase cleavage site between the amino acids LNA and GNI is marked with an asterisk. The underlined part of the amino acid sequences will not be part of the translation if the marked guanidine is removed.</p

Results of the Blastp search for proteases genes, of the localization prediction and calculated molecular weights (MW).

<p>Locus tags and descriptions were taken from the corresponding GenBank entries. The columns “matrix”, “<i>E</i>-Value”, “bitscore” and “% id.” list the alignment data of the according highest scoring alignment. The column “class” refers to our definition of alignment classes. Molecular weight was calculated by a program hosted on the ExPASy server. The columns “CELLO”, “PA-SUB”, and “PSORTb” give the classifications according to the prediction software. The column “Phobius” gives the number of transmembrane helices and a plus (+) sign if a signal peptide was found. Columns “SecP” and “SignalP” contain a plus sign for a SecretomeP output≥0.5 or prediction of a signal peptide, respectively. CP = cytoplasm, IM = inner membrane, PP = periplasm, OM = outer membrane, EX = extracellular, U = unknown.</p

Proteins that were identified by mass-spectrometry (<i>cf.</i>Figure 2B).

<p>^aresults of two independently processed samples; ^bprotein score was below the level that indicates a <i>p</i>-value of <0,05, ^cnot determined.</p

<i>H. pylori</i> secretes bacterial factors with caseinolytic activities.

<p>(A) The <i>H. pylori</i> strain 26695 was grown in protein-free BHI medium. After 48 hours, the bacteria were harvested and lysed by sonfication (<i>Hp</i> son). 30 µl aliquots of the supernatants (BHI <i>Hp</i>) and bacterial lysates were separated by casein zymography and analyzed proteolytic activities. (B) <i>H. pylori</i> strains wild type (P12, wt), ΔPAI, and ΔVacA were grown in protein-free liquid growth medium. 30 µl of the aliquots of the medium were analyzed in casein zymograms for proteolytic activities.</p

Proteolytic activity of the Hp1018/19 protein.

<p>(A) For the construction of the GST-Hp1018/19Δsp fusion protein, the re-sequenced Hp1018/19 gene was amplified without the putative signal peptide and cloned into the pGEX-6P-1 vector. (B) The <i>gst-hp1018/19Δsp</i> construct was transformed in <i>E. coli</i> for overexpression and total protein extracts from untreated (lane 1) and IPTG-induced <i>E. coli</i> (lane 2) were separated by SDS PAGE. Overexpressed GST-Hp1018/19Δsp was precipitated using glutathione sepharose and released by three eluation steps (lanes 3–5). To remove the GST tag, GST-Hp1018/19Δsp bound to glutathione sepharose were treated with PreScission protease and 30 µg protein of the supernatant containing the Hp1018/19 (lane 6) were loaded on a SDS PAGE followed by Coomassie staining. (C) Three µg of purified GST-Hp1018/19Δsp^S205A (lane 1), GST-Hp1018/19Δsp (lane 3), PreScission protease-treated Hp1018/19Δsp^S205A (lane 2) and Hp1018/19Δsp (lane 4) were analyzed by casein zymography for proteolytic activity.</p

Identification of <i>H. pylori</i> proteases.

<p>(A) For a preparative analyses, 18×10⁹ bacteria were lysed and analyzed by zymography. The upper (1) and lower (2) negatively stained protease bands were excised, proteins were eluated and separated by SDS PAGE and Coomassie staining (B). Indicated protein bands were analyzed by mass-spectrometry.</p

Ten validated mutations with the lowest FDRs, selected out of a set of 2396 exonic variations which were found in duplicate in two B16 samples.

<p>None of these mutations is present in dbSNP (version 128; genome assembly mm9).</p

Schematic overview of FDR calculation method.

<p><b>A</b> Concept of FDR calculation which relies on the availability of a normal tissue replication experiment. <b>B</b> Examples of single nucleotide variations found: A somatic mutation found in all three B16 samples (left), a non-somatic mutation found in all B16 and black6 samples (middle) and a mutation found in only one black6 sample (right); this last variation would cause a raise in the FDR for all somatic mutations with a comparable or worse quality. <b>C</b> Process to generate FDRs for a set of somatic mutations and visualize the results. The FDR distribution is visualized as an average estimated ROC curve with the grey bars giving the 95% confidence interval for the mean in both dimensions at uniformly sampled positions. The mean was obtained from the distribution of estimated ROC curves of the FDRs for all possible 18 combinations of reference data sets (see text).</p

Overview of the process for finding somatic mutations in B16.

<p>Numbers for the individual steps are given as an example for one B16 sample, compared to one black6 sample. “Exons” refers to the exon coordinates defined by all protein coding RefSeq transcripts.</p

Comparison of different experimental settings and analysis procedures.

<p><b>A</b> Estimated ROC curves for the comparison of the three different software tools (duplicates, 38× coverage). <b>B</b> Estimated ROC curves for the comparison of different average sequencing depths (SAMtools, no replication). 38× denotes the coverage obtained by the experiment, while other coverages were down sampled starting with this data. <b>C</b> Estimated ROC curves visualizing the effect of experiment replication (38× coverage, SAMtools). <b>D</b> Estimated ROC curves for different sequencing protocols (SAMtools, no replication). The curves were calculated using the results of the 2×100 nt library (<b>Note</b>: A complete display of the results can be found in Supplementary Figures S2 and S3 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002714#pcbi.1002714.s005" target="_blank">Text S1</a>. Also, unscaled versions of the plots are shown in Supplementary Figure S8 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002714#pcbi.1002714.s005" target="_blank">Text S1</a>, giving an impression of the individual set sizes).</p