RNAseq data of blood samples from children with pneumonia, M. pneumoniae pneumonia and Healthy Controls used to validate transcriptomic signatures of M.pneumoniae penumonia

Pneumonia remains the leading cause of death in children under five, but existing diagnostic methods frequently lead to innecessary or mistaken treatment. M. pneumoniae lacks cellular wall so it does not respond to common firs-line antibiotic. Our study aims to guide the diagnosis and treatment by identifying host transcriptomic biomarkers in the blood of children with Mycoplasma pneumoiae pneumonia. Using RNA sequencing, we identified and validated 8 different n-transcript signature that accurately differentiates M. pneumoniae pneumonia from the rest of pneumonias. A strand specific library preparation was completed using NEBNext® Ultra™ II mRNA kit (NEB) and NEB rRNA/globin depletion probes following manufacturer’s recommendations. Individual libraries were normalized using Qubit, pooled together and diluted. The sequencing was performed using a 150 or 75 paired-end configuration in a Novaseq6000 or HiSeq 4000 platforms. Quality control of raw data was carried out using FastQC, alignment and read counting were performed using STAR, alignment filtering was done with SAMtools and read counting was carried out using FeatureCounts. RNAseq data was processed for batch correction using control samples and COMBAT-Seq package.