Computational protocol: Sequential inflammatory processes define human progression from M. tuberculosis  infection to tuberculosis disease

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Protocol publication

[…] Generation of the whole blood RNA-Seq data was previously described []. RNA was extracted from PAXgene tubes, globin transcripts were depleted and (GlobinClear, Life Technologies) cDNA libraries were prepared using Illumina mRNA-Seq Sample Prep Kit. RNA-Seq was performed by Expression Analysis Inc., at 30 million 50bp paired-end reads, on Illumina HiSeq-2000 sequencers.For monocytes and T cells, RNA was extracted from cells left unstimulated, stimulated with M.tb antigens (ESAT-6/CFP-10 or Ag85A/B; T cells), or infected with M.tb (monocytes and T cells). RNA-Seq was performed by Expression Analysis Inc. as described [] or (Unstimulated and M.tb antigen-stimulated T cells) Beijing Genomics Institute (Shenzen, China) after performing amplification (Clontech SMARTer Universal Low Input RNA Kit). RNA-Seq alignment, QC, and gene-level summarization for whole blood, monocytes, and T cells were also performed as described [].Whole blood, monocyte and CD4 T cell RNA-Seq data was aligned to the hg19 human genome using gsnap [] as in the original study []. Normalized gene-level expression estimates were derived from mapped read pairs following the procedure implemented previously []. Briefly, mapped read pairs were assigned to genes by collapsing all transcripts into a single gene model and counting the number of reads that fully overlap the resulting exons using htseq (v. 0.6.0) [], with strict intersection and including strand information. Gene models for protein-coding genes were downloaded from Ensembl (GRCh37.74). Reads that mapped to multiple locations were only counted once and those mapping to ambiguous regions were excluded. Log2-transformed values of counts normalized by adjusted library counts were computed using the cpm function of the edgeR package []. For monocyte and CD4 T cell transcriptomic analyses, both RNA-Seq and qRT-PCR measurements of the ACS signature of risk of TB score () were used to classify samples as positive (> 0.6 in both RNA-Seq and qRT-PCR) or negative (< 0.4 in both RNA-Seq and qRT-PCR), to ensure robust classification. […]

Pipeline specifications

Software tools GSNAP, HTSeq, edgeR
Application RNA-seq analysis
Organisms Homo sapiens, Mycobacterium bovis BCG
Diseases Tuberculosis, Tuberculosis, Pulmonary