Figure S1. Outline of the dual-color RT-MLPA assay.

Dual-color RT-MLPA is based upon the ligation of two chemically synthesized half-probes when hybridized adjacently to a target sequence. Ligation was facilitated by 5’ phosphorylation of the right hand half-probe. Fragments were amplified with the use of only two fluorophor-conjugatedPCR primer pairs while the discriminative length of each amplicon was assured by varying the length of the target-specific sequence in each probe set. The length increased in a stepwise fashion by only 2 nucleotides, with a total size range lying between ~80-220 nucleotides. This size range provided optimal fragment separation and a low background using capillary electrophoresis. Fluorescence-intensity of each amplification product (peak area) was quantified using GeneMapper software package and category tables containing peak areas were exported for further analysis.

Figure S2. Identification of individual biomarkers that are differentially expressed between TB cases, LTBIs, and uninfected healthy controls.

Biomarkers differentially expressed between TB cases, LTBIs, and uninfected healthy controls were identified by pairwise testing of the data set using the Global Test followed by multiple testing-correction. P-values indicate the strength of the association between the individual biomarkers and the groups tested, while the hierarchical-clustering tree above each bar-diagram shows the absolute correlation between the biomarkers analyzed, with the part of the tree that is significant after multiple testing correction colored dark, and the part that is non-significant after correction in gray. The color of the bar indicates in which study group that particular gene is more abundantly expressed.

Figure S3. Correlation between Paxgene tube storage conditions and quality of purified RNA.

PAXgene tubes were stored after venipuncture blood collection at different temperatures and total RNA was extracted (yielding approximately 4-5 µg of RNA) at the indicated timepoints. To assess the quality and integrity of the RNA, samples were run on an Agilent 2100 BioAnalyzer using the RNA 6000 Nano Chip kit. The average RIN (RNA integrity number) of the total RNA samples is displayed in the left panel and the concomitant gel-like graphical view is shown in the right panel. PAXgene tubes can be stored at room temperature for up to 24 hours (data not shown), at 4ºC for up to a month and long-term at -20ºC to -80ºC.

Figure S4. R script used to perform lasso regression analysis.

Table S1. Sequences of RT-primers and half-probes comprising the complete dcRT-MLPA probe set.

Indicated are the terminal priming sequences that flank the half-probes and are recognized by the HEX-labeled MAPH primers (green) and FAM-labeled MLPA primers (blue) for PCR amplification.

Table S2. Identification of individual biomarkers that are differentially regulated between TB cases, LTBIs, uninfected healthy controls, and during treatment of TB patients.

Dual-color RT-MLPA was performed on direct ex vivo RNA isolated from PAXgene tubes derived from TB cases, LTBIs, and uninfected healthy controls (a) or TB patients treated for 2, 4, or 6 months (b). Relative gene expression levels (peak areas normalized to GAPDH and log2 transformed) of the indicated genes and the median expression within each group was calculated. Significant differences between study groups were determined using Kruskal-Wallis H and Dunn's multiple comparison test. Only genes whose expression levels displayed significant differences between TB cases and LTBIs (a) were further analyzed during treatment of TB patients (b). ND = Not Detected.

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