Domain discovery.

<p>A. Genes categorized by domain-level resolution of regional requirement. B. Genes categorized as containing only required regions (blue), containing both required and non-required regions (navy) and containing no required regions (yellow) were assessed for requirement along the entire length of the gene, creating a single p-value describing the statistical underrepresentation of insertion reads within the whole gene. For each category, the number of genes across the range of p-values are plotted. C. For genes with both required and non-required regions, the likelihood that the relative position within the gene resides in a required region.</p

Transposon junction sequencing accurately reflects true library content.

<p>A. A Mtb mutant library is created by phage-delivery of transposons, disrupting each genome with a single insertion. Shown is a schematic of 6 mutant chromosomes spanning three genes (A–C), with transposons—red arrows—disrupting one of the three genes. After growing the library on 7H10 media, we pooled surviving mutants. In this schematic, gene C is required for optimal growth and thus mutants with transposons in gene C are lost. We isolated genomic DNA from the survivors for transposon site mapping. B. We sheared the genomic DNA by sonication, and repaired frayed ends to create blunt ends. We then used Taq polymerase to generate A-tails, allowing the ligation of T-tailed adapters. Finally, we selectively amplified transposon junctions using primers recognizing the transposon end and the adapter. Primers used for amplification contain all requisite sequences to permit direct sequencing of amplicons on an Illumina Genome Analyzer 2. C. We created a library of identified transposon insertion mutants in known relative quantities. DNA from the library was prepared for transposon junction sequencing. Insertion counts were plotted against the known relative quantity of the mutant in the library. D. To further confirm that read counts were a representation of the number of genomes in the library, we estimated the number of PCR template molecules. For each gene, we plotted the estimate of template molecule count against the read counts.</p

RNAs required for growth <i>in vitro</i>.

<p>A. IGV plot for genomic region containing the rnpB, the RNA component of RNaseL. Tracks, from top to bottom: 1. Histogram of insertion counts, 2. Comprehensive heat-map of requirement of 500-bp windows, 3. Position of annotated genes, 4. Position of TA dinucleotide sites, 5. Position of rnpB. B. IGV plot for genomic region containing the tmRNA. Tracks, from top to bottom: 1. Histogram of insertion counts, 2. Comprehensive heat-map of requirement of 500-bp windows, 3. Position of annotated genes, 4. Position of TA dinucleotide sites, 5. Position of the tmRNA.</p

Functional requirement testing and mapping.

<p>A. Required regions were defined as regions with a statistical underrepresentation of insertion counts compared to the rest of the genome. To test this, we applied a non-parametric test for regions of increasing size, as described in B. Every 250, 400, 500, and 600 bp region (large enough for statistical power) was tested for insertion count underrepresentation, generating a comprehensive map of required regions in the Mtb genome. Tracks on the circularized genome, from inner-most to outer-most: 1. Histogram of insertion counts, 2. Annotated genes, forward direction, 3. Annotated genes, reverse direction, 4. All required regions. 5. Required intergenic regions.</p

<i>ppm1</i> and <i>fhaA</i> each code for two domains with varying requirements for growth.

<p>A. IGV plot for genomic region containing <i>ppm1</i>. Tracks, from top to bottom: 1. Histogram of insertion counts, 2. Comprehensive heat-map of requirement of 500-bp windows, 3. Position of annotated genes, 4. TA sites, 5. Position of known domains within <i>ppm1</i>. B. PCR footprinting for insertions was performed using primers against the an upstream genomic region and the transposon end, resulting in amplicons spanning <i>ppm1</i> to various inserted transposons. Lad: 1 kb DNA ladder, 1: wt Mtb transposon library, 2: <i>ppm1</i>-complemented Mtb transposon library. C. IGV plot for genomic region containing <i>ppm1</i>. Tracks, from top to bottom: 1. Histogram of insertion counts, 2. Comprehensive heat-map of requirement of 500-bp windows, 3. Position of annotated genes, 4. TA sites. D. PCR footprinting for insertions was performed using primers against the an upstream genomic region and the transposon end, resulting in amplicons spanning <i>fhaA</i> to various inserted transposons. Lad: 1 kb DNA ladder, 1: wt Mtb transposon library, 2: <i>fhaA</i>-complemented Mtb transposon library.</p

Con-ARTIST decodes intergenic regions of conditional essentiality.

<p>The <i>vc2635</i> locus, which encodes penicillin-binding protein 1A, was defined as conditionally essential <i>in vivo</i>, while an upstream intergenic region, <i>IG_vc2635</i>, was found to be domain conditionally essential by Con-ARTIST. The reads at each insertion in the <i>in vitro</i> and <i>in vivo</i> grown libraries are shown in orange and all potential transposon insertion sites (TA dinucleotides) are in black. The probabilities for each insertion being conditionally essential (CE, blue) or non-essential (NE, purple) <i>in vivo</i> (no probabilities for conditional enrichment or essential for <i>in vitro</i> growth were found) are overlaid on the locus. Previously published RNAseq data <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004782#pgen.1004782-Mandlik1" target="_blank">[23]</a> was also overlaid on the locus (green). The part of the intergenic region identified as conditionally essential overlaps with the predicted −10 and −35 boxes (yellow box with arrow) and putative 5′ UTR sequence. The region presented shows reads spanning nucleotides 2805484 to 2806320 (∼800 bases).</p

Con-ARTIST improves detection of conditionally essential genes compared to previous studies.

<p>(A) Overlap of the conditionally essential genes in <i>M. tuberculosis</i> required for mouse infection as determined by Con-ARTIST, Sassetti et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004782#pgen.1004782-Sassetti1" target="_blank">[15]</a> and Zhang et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004782#pgen.1004782-Zhang1" target="_blank">[7]</a>. The overlap between Con-ARTIST and Sassetti et al. was significantly better than the overlap between Zhang et al. and Sassetti et al. (p-val<0.05, one-tailed Fisher's exact test). (B) The standard deviations of p-values across 100 simulation-based MWU tests were plotted for either Con-ARTIST conditionally essential genes in <i>M. tuberculosis</i> that overlap with Zhang et al. (75 genes), or for genes that were significant only in the Zhang et al. dataset (267 genes). In all three mice, genes that overlap between Con-ARTIST and Zhang et al. had significantly (*, p-value<0.0005) narrower ranges of p-value standard deviations across MWU tests than genes categorized as conditionally essential only by Zhang et al. (C) Overlap of conditionally essential genes required for <i>V. cholerae</i> rabbit infection as defined by Con-ARTIST, Kamp et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004782#pgen.1004782-Kamp1" target="_blank">[16]</a> or Fu et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004782#pgen.1004782-Fu1" target="_blank">[11]</a>. Genes that were defined as defective for <i>in vitro</i> growth by Kamp et al. were filtered from both the Con-ARTIST and Fu et al. results. The overlap between Con-ARTIST and Kamp et al. was significantly higher than the overlap between Kamp et al. and Fu et al. (p-value<0.0001 by one-tailed Fisher's exact test). (D) In-frame deletions were constructed in several <i>V. cholerae</i> genes that were either identified as required for rabbit infection in previous studies (but not by Con-ARTIST) or unique predicted to be conditionally essential (CE) in this study. WT and mutant cells were first barcoded and then pooled to infect rabbits. The ratios of each mutant barcode compared to WT sequences were compared before and after infection to generate a competitive index. The competitive indexes for each mutant are coordinately colored according to the individual animal from which they were derived. One outlier measurement was identified (in <i>Δvc0432</i>) using the Grubbs test and removed. *, significantly underrepresented, p-value≤0.01 by Kruskal-Wallis test, with Dunn's test for multiple comparisons.</p

Sequence enrichment profiles after high-throughput sequencing of the phage displayed libraries using Illumina technology.

<p>(A) Number of unique peptides observed in the different rounds of biopanning (B) Frequency of the selected peptides at each round of biopanning (C) Manhattan plot showing peptide sequence enrichment (GWAS) results for round 3 and 5 of biopanning.</p

Con-ARTIST reduces false positive assignments.

<p>(A) Two simulated libraries derived from the same <i>M. tuberculosis</i> control dataset were compared against each other either using MWU analysis alone, or the full Con-ARTIST pipeline. We used a range of p-value cutoffs as the thresholds for defining whether an insertion site is being called significantly different in reads. The number of insertions that are called significantly different (i.e., being false positively assigned) between simulations when using a range of p-value cutoffs was then determined. (B) <i>In vitro</i> grown and animal infection datasets from <i>M. tuberculosis</i> and <i>V. cholerae</i> were run through the Con-ARTIST pipeline. The <i>in vitro</i> datasets from each organism were also analyzed by EL-ARTIST. The concordance of insertion sites similarly defined as essential for growth <i>in vitro</i> across 100 simulations was compared between the arms for both pathogens.</p

Con-ARTIST identifies conditionally essential regions required for host infection at sub-genic resolution.

<p>Con-ARTIST identified domain-encoding regions in <i>M. tuberculosis rv0018c</i> (A) and <i>V. cholerae vc2041</i> (B) that exhibit conditional essentiality <i>in vivo</i>. Reads in each organism (blue and orange) from <i>in vitro</i> and <i>in vivo</i> grown transposon libraries were mapped to each potential insertion site (TA dinucleotides, black bars) in the genome. Protein domains predicted by Pfam (black lines) and transmembrane segments predicted by Phobius (red boxes) are shown. The Con-ARTIST probabilities for each insertion being conditionally essential (blue), essential for growth <i>in vitro</i> (red) or non-essential <i>in vivo</i> (purple) are graphed along the gene (no probabilities for conditional enrichment were detected). The cellular compartments of Rv0018c (C) and VC2041 (D) protein domains were predicted and overlaid with the predicted Pfam protein domains (black lines) using Protter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004782#pgen.1004782-Omasits1" target="_blank">[36]</a>. Con-ARTIST-defined regions of essentiality (pink shaded region) and conditionally essentiality (blue shaded region) were also overlaid. <i>rv0018c</i> covers ∼1.5 kb, while vc2041 is ∼1.8 kb.</p