Mtb Datasets
TnSeq Datasets
General Notes:
- libraries were made with Himar1 transposon using the protocol described in Long et al. (2015)
- reads were mapped to TA dinculeotide sites using BWA, and TPP in Transit
- insertion counts provided in .wig or combined_wig file format: column 1 gives coordinates of TA sites (relative to the parental strain/reference genome); individual samples/replicates are in subsequent columns
- conditionally essential genes determined by 'resampling' in Transit; hits are defined as Qval<0.05
- most of the data files are in tab-separated .txt format to be opened as spreadsheets; header/comment lines are prefixed with '#'
TnSeq: Reference list of essential genes for in-vitro growth of Mtb (DeJesus et al, 2017)
- Summary: A high-saturation dataset was developed by combining 14 independent Tn libraries in H37Rv. The data was analyzed by a Hidden Markov Model (HMM), to identify 4 categories of genes: essential (ES), growth-defect (GD), non-essential (NE), and growth-advantaged (GA).
- Reference: DeJesus MA, Gerrick ER, Xu W, Park SW, Long JE, Boutte CC, Rubin EJ, Schnappinger D, Ehrt S, Fortune SM, Sassetti CM, Ioerger TR. Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis. mBio. 2017 Jan 17;8(1):e02133-16. doi: 10.1128/mBio.02133-16. PMID: 28096490; PMCID: PMC5241402.
- raw sequencing data on SRA: PRJNA341349
- insertion counts: 14_replicates_combined_wig.txt (reference genome = H37RvBD = NC_018143)
- gene-level essentiality calls (490 ES+135 GD genes) determined by HMM: Table S3
TnSeq: Genes required for Mtb growth on cholesterol (Griffin et al., 2011)
TnSeq of PonA1 knockout (Kieser et al., 2015)
- Summary: Knockouts of peptidoglycan synthesis genes: Rv0050/PonA1, Rv3682/PonA2, and Rv2518c/LdtB
- Reference: Kieser KJ, Baranowski C, Chao MC, Long JE, Sassetti CM, Waldor MK, Sacchettini JC, Ioerger TR, Rubin EJ. Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility. Proc Natl Acad Sci U S A. 2015 Oct 20;112(42):13087-92. doi: 10.1073/pnas.1514135112. Epub 2015 Oct 5. PMID: 26438867; PMCID: PMC4620856.
- insertion counts: PonA1.combined_wig.txt, PonA1.combined_wig.txt (reference genome = H37Rv)
- conditional essentials by resampling:
- sequencing data on SRA: SRA277968
- PATRIC link
TnSeq of Mtb grown in hypoxic conditions (Rittershaus et al., 2018)
- Summary: H37Rv Tn library grown in sealed roller bottles for 3 and 6 weeks.
- Reference: Rittershaus ESC, Baek SH, Krieger IV, Nelson SJ, Cheng YS, Nambi S, Baker RE, Leszyk JD, Shaffer SA, Sacchettini JC, Sassetti CM. A Lysine Acetyltransferase Contributes to the Metabolic Adaptation to Hypoxia in Mycobacterium tuberculosis. Cell Chem Biol. 2018 Dec 20;25(12):1495-1505.e3. doi: 10.1016/j.chembiol.2018.09.009. Epub 2018 Oct 11. PMID: 30318462; PMCID: PMC6309504."
- sequencing data: data not on SRA
- insertion counts: hypoxia_combined_wig.txt, hypoxia_metadata.txt, parental strain=H37Rv
- 268 conditionally essential genes by resampling (at 6 weeks vs. Input): resampling_hypoxia_6wks.xlsx
TnSeq of Rv0060/DarG knockdown (Zavari et al, 2020)
- Summary: Rv0059/60 are a toxin/antitoxin pair (ADP-ribosyl glycotransferase and hydrolase). Rv0060 was depleted to 4 levels via Clp-mediated degradation (sspB expression controlled by Tet-ON promoter induced with 500, 50, 25, and 15uM ATC)
- Reference: Zaveri A, Wang R, Botella L, Sharma R, Zhu L, Wallach JB, Song N, Jansen RS, Rhee KY, Ehrt S, Schnappinger D. Depletion of the DarG antitoxin in Mycobacterium tuberculosis triggers the DNA-damage response and leads to cell death. Mol Microbiol. 2020 Oct;114(4):641-652. doi: 10.1111/mmi.14571. Epub 2020 Jul 28. PMID: 32634279; PMCID: PMC7689832.
- insertion counts: teton_Rv0060_combined_wig.txt, teton_Rv0060_samples_metadata.txt (reference genome: H37RvMA2)
- 60 conditionally essential genes: resampling_15_vs_500.pooled.xlsx
- sequencing data on SRA: PRJNA640933
TnSeq: Antibiotic tolerance (Xu et al. 2017)
- Summary: H37Rv Tn library exposed to sub-MIC concentrations of isoniazid (INH), rifampicin (RIF), meropenem (MERO), Vancomycin (VANC), and Ethambutol (EMB).
- Reference: Xu W, DeJesus MA, Rücker N, Engelhart CA, Wright MG, Healy C, Lin K, Wang R, Park SW, Ioerger TR, Schnappinger D, Ehrt S. Chemical Genetic Interaction Profiling Reveals Determinants of Intrinsic Antibiotic Resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2017 Nov 22;61(12):e01334-17. doi: 10.1128/AAC.01334-17. PMID: 28893793; PMCID: PMC5700314.
- insertion counts: Abx_combined_wig_TTR.txt, Abx_samples_metadata.txt (reference genome=H37Rv)
- 251 genes conditionally essential in at least 1 of the 5 drugs: Table S1
- sequencing data on SRA: PRJNA436485
TnSeq: Genes required for growth in acidic conditions (pH 5.5)
TnSeq in C57BL/6 mice (animal model of infection)
- Summary: 6 high-quality replicates in-vivo (passaged through C57BL/6 mice) vs 6 replicates in-vitro.
- Reference: Subramaniyam S, DeJesus MA, Zaveri A, Smith CM, Baker RE, Ehrt S, Schnappinger D, Sassetti CM, Ioerger TR. Statistical analysis of variability in TnSeq data across conditions using zero-inflated negative binomial regression. BMC Bioinformatics. 2019 Nov 21;20(1):603. doi: 10.1186/s12859-019-3156-z. PMID: 31752678; PMCID: PMC6873424.
- data collected by Clare Smith (Sassetti lab, UMMS)
- raw sequencing data: not on SRA
- insertion counts: IV_combined_wig_TTR.txt, IV_samples_metadata.txt (reference genome=H37RvBD1)
- 237 conditionally essential genes by ZINB analysis: Supp Table 1
TnSeq: Genes required for survival of infections in mice with immune deficiencies (knockout mice: MHCII-/- and CD4-/-) (Zhang et al., 2013)
- Summary: This was a large-scale study of H37Rv infections in mice with immune deficiencies. A Tn library of H37Rv was
passaged through MHCII-/- and CD4-/- knock-out mice, and compared to C57BL6 mice,
as well as several surrogate in-vitro stress conditions (nitric oxide, low pH, high iron) for comparison.
The study led to the identification of the importance of the tryptophan biosynthesis pathway during infection.
- Reference: Zhang YJ, Reddy MC, Ioerger TR, Rothchild AC, Dartois V, Schuster BM, Trauner A, Wallis D, Galaviz S, Huttenhower C, Sacchettini JC, Behar SM, Rubin EJ. Tryptophan biosynthesis protects mycobacteria from CD4 T-cell-mediated killing. Cell. 2013 Dec 5;155(6):1296-308. doi: 10.1016/j.cell.2013.10.045. PMID: 24315099; PMCID: PMC3902092.
- insertions counts: Table S4, jason_orig_combined_wig.txt, jason_orig_metadata.txt (reference genome = H37RvBD1)
- raw sequencing data: not on SRA
- conditions:
- in vitro control, and input library (for mouse infections)
- wt mouse d10, d45
- MHCII-/- mouse d10, d45
- CD4-/- d45
- Fe 450 uM, 1.5 mM
- pcit pH 4.5 (phosphate/citrate buffer)
- Tyloxapol pH 4.5, 6.5, 7.0
- DETA-NO (nitric oxide; use Tylox-pH7 as control)
- Trp Rescue
- AA Rescue
TnSeq: Knock-outs of virulence genes (Rv0307c, Rv1432, and Rv2680) passaged through mice to determine genetic interactions (DeJesus et al., 2017)
- Summary: Tn libraries of genetic knock-outs of Rv0307c, Rv1432, and Rv2680 were passaged through mice to identify genetic interactions (other genes required in their absence, specifically in-vivo). These 3 genes were selected because they had previously been found to be virulence genes, required for survival in-vivo but not in-vitro, though their functions are unknown. Each library was profiled at day0 and day32 in C57BL/6 mice, and compared to a wild-type library (H37Rv). The data (2x2=4 conditions for each gene) was analyzed using a Bayesian method ('GI' method in Transit) to quantify the statistical significance of interacting genes (genes that exhibit conditional essentiality in the KO (compared to WT) that is specific to the in-vivo condition (compared to in-vitro).
- Reference: DeJesus MA, Nambi S, Smith CM, Baker RE, Sassetti CM, Ioerger TR. Statistical analysis of genetic interactions in Tn-Seq data. Nucleic Acids Res. 2017 Jun 20;45(11):e93. doi: 10.1093/nar/gkx128. PMID: 28334803; PMCID: PMC5499643.
- data collected by Subu Nambi (Sassetti lab, UMMS)
- raw sequencing data on SRA: PRJNA338716
- insertion counts: subu_combined_wig.txt, subu_samples_metadata.txt (reference genome=H37Rv)
- Results: Table S2, Rv1432-KO: 35 genetic interactions, Rv2680-KO: 75 genetic interactions, Rv1565c-KO: 83 genetic interactions
TnSeq: Mtb knock-out strains of ESX-1 genes (EccD1, EspI, PE35, and PPE68)
- Summary: TnSeq of Mtb knock-out strains of EccD1, EspI, PE35, and PPE68 (in the ESX-1 locus) compared to H37Rv_R96 as control, grown in-vitro (7H9), 1 replicate each.
- Reference: unpublished
- data collected by Rebecca Audette (Eric Rubin lab, HSPH)
- raw sequencing data: PRJNA484233
- insertion counts: ESX1_combined_wig.txt, ESX1_metadata.txt, (reference genome=H37RvMA2)
- resampling output files: resampling_EccD1_KO.xlsx, resampling_EspI_KO.xlsx, resampling_PE35_KO.xlsx, resampling_PPE38_KO.xlsx
- conditionally essential genes by resampling: EccD1: 7, EspI: 9, PE35: 5, PPE68: 4
TnSeq: Mtb knock-out strains of peptidoglycan hydrolases (Rv3717/ami, Rv3594/ami4 and Rv3811/ami3)
TnSeq: Mtb knock-out strains of more peptidoglycan hydrolases (Rv0950, Rv1096, Rv3684)
TnSeq: Mtb knock-out strain of Rv3671c/MarP
TnSeq: Mtb knock-out strain of Rv0954
RNAseq Datasets
General Notes
- DEGs = Differentially Expressed Genes
- used DeSeq2 for analysis - significant genes are those with adjusted Pval<0.05 and |LFC|>1.0.
RNAseq: Differential gene expression in knock-out of Rv3263/MamA (Shell et al., 2013)
RNAseq: Genes with periodic expression over the Mtb cell cycle (Bandekar et al., 2020)
RNAseq: Differential expression in knock-out strains of Rip1 and Rv3193c in iron-limiting conditions
- Summary: Knock-outs of Rip1 and Rv3193c cause sensitivity to iron limitation. In this experiment,
a KO of Rip1 was made in the Erdman strain of Mtb, and a KO of Rv3193c was made in the H37Rv strain.
Phenanthroline was used as a chelator to deplete iron from the 7H9 medium.
- Reference: not published (data collected by Sami Nelson in Sassetti lab, circa 2015)
- parental strains: Mtb Erdman, H37Rv
- data on GEO: GSE67670
- counts: GSE67670_Rip1-RNA-Seq-counts.xlsx, RPKMs: GSE67670_Rip1-RNA-Seq-RPKMs, metadata: Rip1-conditions.txt
- DeSeq analysis:
RNAseq: Differential expression in knock-out strains of Rip1 and SigL in iron-limiting conditions (Nelson et al, 2023)
- Summary: Knock-outs of Rip1 and SigL cause sensitivity to iron limitation. In this experiment,
a KO of Rip1 and SigL was made in the H37Rv strain of Mtb.
Dipyridyl (DPD; 50 and 100 uM) was used as a chelator to deplete iron from the 7H10 medium.
- Reference:
Nelson SJ, Williams JT, Buglino JA, Nambi S, Lojek LJ, Glickman MS, Ioerger TR, Sassetti CM.
The Rip1 intramembrane protease contributes to iron and zinc homeostasis in Mycobacterium tuberculosis. mSphere. 2023 Aug 24;8(4):e0038922.
doi: 10.1128/msphere.00389-22. Epub 2023 Jun 15. PMID: 37318217; PMCID: PMC10449499.
- data on GEO: GSE229446
- metadata: Rip1_SigL_samples_metadata.xlsx, RPKMs: Rip1_SigL_RPKMs.xlsx
- samples also included KO of Rv3193c, along with Rip1 and SigL
- exclude sample #29? (supposed to be Rip1 KO, but shows expression)
- DeSeq analysis (DEGs)
- Genetic Interaction analysis (using GI in Transit)
DNA Microarray: Transcriptional Responses to Drugs
RNAseq: Identification of Transcriptional Start Sites (TSS) in Mtb H37Rv
- Summary: RNA extracted from log-phase cultures was extracted and sequenced
with and without polyphosphatase treatment to hydrolyze 5' triphosphate caps on transcripts.
TSSs were identified by sites with significant differences in apparent expression levels.
Most TSSs were proximal to translational
start sites, and a large fraction (~1/3) of transcripts were found to be leaderless,
where transcriptional and translational start sites co-occur, lacking a 5'UTR.
- Reference:
Shell SS, Wang J, Lapierre P, Mir M, Chase MR, Pyle MM, Gawande R,
Ahmad R, Sarracino DA, Ioerger TR, Fortune SM, Derbyshire KM, Wade
JT, Gray TA. Leaderless Transcripts and Small Proteins Are Common
Features of the Mycobacterial Translational Landscape. PLoS
Genet. 2015 Nov 4;11(11):e1005641. doi:
10.1371/journal.pgen.1005641. PMID: 26536359; PMCID: PMC4633059.
- sequencing data (raw fastq files) on GEO: GSE62152
- coordinates of start sites: Table S3 (relative to genome NC_000962)
- operons predicted based on TSSs: TSS_operons_scarlet.txt
RNAseq: Differential gene expression of H37Rv in stress conditions; and rpoB mutants of a Beijing strain
- collected in lab of Sarah Fortune, HSPH (circa 2013)
- Reference: not published
- data on GEO: GSE67035
- conditions:
- H37Rv on defined carbon sources: 4G) 4% glucose, 1B) 1% butyrate, or 2G1B) 2% glucose+1% butyrate (in minimal medium)
- H37Rv in low-vs-high iron: HiFe) high Fe, LoFe_1D) low Fe 1 day, LoFe_1W: low Fe 1 week (in 7H9)
- H37Rv in acid: pH 5.5 vs. pH 7.0 (in 7H9 with tyloxapol)
- Beijing strain and rpoB mutants: a) WT rpoB, b) rpoB:D516, c) rpoB:H526D, d) rpoB:H526R, e) rpoB:S531L
- U19_RNAseq_metadata.xlsx, U19_expr_RPKMs.xlsx, U19_RNAseq_counts.xlsx
- DEGs by DeSeq2
RNAseq: Differentially expressed genes in Mtb gene knock-out (KO) strains (U19)
- Summary: knock-outs of the following genes, compared to H37Rv (parental strain) grown in 7H9:
- Rv2680, Rv1096, Rv1565c, Rv3484, Rv3684, Rv3916c, Rv0307c, Rv1432, Rv0950c
- data collected by Shoko Wakabayashi in lab of Eric Rubin (HSPH) (2018)
- Reference: unpublished
- data on GEO: GSE134574
- DeSeq2 files with DEGs:
Metabolomic Datasets
Genomes and Annotations
- these files are needed for doing various analysis in Transit
- the genomes have slightly different sequences and ORF coordinates
- H37Rv: genome, prot_table Cole et al (NC_000962.2)
- H37Rv3: genome, prot_table updated (NC_000962.3)
- H37RvMA2: genome, prot_table - strain used in several labs (GCF_000751615.1), re-sequenced, has a few genetic differences
- H37RvBD1: genome, prot_table - re-sequenced at Broad Institute (NC_018143.1), with annotations based on H37Rv