High concordance of biological replicates.

Pairwise distance values calculated from variance stabilizing transformed expression counts between samples that are not biological replicates (NO) and samples that are biological replicates (YES). Biological replicates have significantly shorter distances (Mann Whitney U Test, p = 3e-5) than non biological replicates with two outliers: MT31 evolved planktonic (*) and MT72 ancestral biofilm (**) samples show discordance between their biological replicates which we took into consideration in the analysis of our differential expression results. (PNG)</p

S3 Fig -

A) Principal component analysis (PCA) of variance stabilizing transformed gene expression counts from ancestral populations. Each point represents the total gene expression of a single sample, where the shape indicates the growth condition of the sample, and the color indicates the population. B) PCA (same as in A) colored this time by biofilm wet weight. There is no clear correlation–either among biofilm samples or planktonic samples–between gene expression patterns and ancestral biofilm phenotype (as measured by wet weight). (PNG)</p

Genetic background shapes adaptive trajectories.

A) Principal component analysis (PCA) of variance stabilizing transformed expression counts from ancestral and evolved biofilm populations. Arrows are drawn between corresponding ancestral and evolved populations, indicating the trajectory of evolution across passaging. Points are colored by sub-lineage of the ancestral population. B) PCA of variance stabilizing transformed expression counts from ancestral and evolved populations grown under planktonic conditions. Like panel A, arrows are drawn between corresponding ancestral and evolved populations and are colored by sub-lineage. Biofilm passaging reduced transcriptome diversity, with populations converging on a sub-lineage-specific signature (A); this pattern was not observed under planktonic growth conditions (B). N.B. While the positions of ancestral and evolved states are known (dots on the PCA), the trajectories between them are not, and connecting lines shown here are schematic. C) Bar plot of DEGs comparing evolved and ancestral biofilm populations broken down by sub-lineage. There are 680 DEGs shared between sub-lineages, and 405 and 200 DEGs unique to L4.4 and L4.9, respectively. Opacity of the bar indicates the subset of DEGs that are either up (dark) or downregulated (light) after passaging. The pellicle biofilm transcriptome is characterized by broad scale downregulation of gene expression with a smaller complement of genes that are upregulated in a sub-lineage specific manner. D) Differential expression of DEGs unique to each sub-lineage, from the comparison of evolved to ancestral biofilm populations. Log transformed adjusted p-values plotted against the log2 fold change for each gene. Genes that did not have significant differential expression are shown in grey. Lineage 4.9 evolved a larger complement of upregulated genes in response to biofilm passaging.</p

Most significantly downregulated (< -5 L2FC) and upregulated (> 2 L2FC) ncRNAs in evolved populations grown as biofilms (compared to planktonic cultures).

Features included in this list are significantly differentially expressed when all populations were analyzed together, as well as in at least five individual populations. Log2 fold change (L2FC) values given for analysis of all populations together.</p

Expression of ncRNAs and sORFs mirror coding regions after passaging.

A) Volcano plot summarizing differential expression of ncRNAs and sORFs in evolved populations. Log transformed adjusted p-values plotted against the log2 fold change for each gene. Genes that did not have significant differential expression are shown in grey. B) Heatmap of normalized, variance stabilizing transformed expression counts for ncRNAs and sORFs with significant differential expression in evolved populations. Each column is a single sample from an evolved population, grown either as a biofilm or in a planktonic culture. Each row is a gene labeled either ncRNA or sORF. Expression values for each gene are normalized to the mean across samples. Samples are clustered by Euclidean distance and plotted as a tree at the top of the heatmap. C) Principal component analysis (PCA) of variance stabilizing transformed expression counts of ncRNAs and sORFs from ancestral and evolved biofilm populations. Arrows are drawn between corresponding ancestral and evolved populations, indicating the trajectory of evolution across passaging. Points are colored by sub-lineage of the ancestral population. The effects of biofilm passage on ncRNAs and sORFs mirror that of coding regions: reduced transcriptome diversity and widespread downregulation with populations converging on a sub-lineage-specific signature. N.B. While the positions of ancestral and evolved states are known (dots on the PCA), the trajectories between them are not, and connecting lines shown here are schematic.</p

No batch effects observed for library preparation or sequencing batch.

Principal component analysis (PCA) of variance stabilizing transformed gene expression counts for all samples, colored by library preparation batch (A) or sequencing batch (B). Resequencing refers to additional sequencing of some samples to achieve target sequencing depth. (PNG)</p

S9 Fig -

A) Log-2 fold changes (L2FC) for all genes in each biofilm population, separated by their presence in one or both duplications acquired by MT31 and MT55. Mann Whitney U test with Benjamini-Hochberg correction shows significantly higher L2FC values for duplicated genes in MT31 and MT55 (same data shown in Fig 6C), and significantly lower L2FC values for duplicated genes in MT49 and MT72. Comparison is between evolved populations grown as a biofilm and ancestral populations grown as biofilms. B) Same as A, but comparing pellicle evolved populations grown as planktonic cultures, to ancestral populations grown as planktonic cultures. Mann Whitney U test with Benjamini-Hochberg correction shows significantly higher L2FC values for duplicated genes in MT31 and MT55 and significantly lower L2FC values for duplicated genes in MT49. (PNG)</p

DEGs comparing pellicle passaged (evolved) populations grown as biofilms, to ancestral populations grown as biofilms.

DEGs provided for multiple analyses: all populations, each population individually and for each of the two sub-lineages in our sample (L4.4 and L4.9). Note that low concordance between biological replicates of evolved MT31 grown under planktonic conditions may impact the results of these analyses. (XLSX)</p

Protocol for the serial passage of <i>M</i>. <i>tb</i> pellicle biofilms and subsequent transcriptomic comparisons.

Ancestral (clinical) populations were grown in planktonic cultures, then grown as pellicle biofilms (passage 1) before passaging 8–12 times. This passaged (evolved) population was taken from a biofilm culture and grown planktonically again. We performed RNA sequencing on all ancestral and evolved populations, grown under both biofilm and planktonic conditions.</p

Heatmaps of variance stabilizing transformed expression counts all genes in the genome.

Each column is a single sample from an ancestral (A) or evolved (B) population, grown either as a biofilm or in a planktonic culture. Each row is a gene. Heatmap colored by expression values for each gene which are normalized to the mean across samples. Samples are clustered by Euclidean distance and plotted as a tree at the top of the heatmap. Evolved populations have much more uniform biofilm transcriptomes. (PNG)</p