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To the Editor: The emergence of new massive sequencing methods has proved revolutionary for the study of complex microbial populations such as the microbiota of the respiratory tract in patients with chronic obstructive pulmonary disease (COPD) [1]. Using this powerful methodology, our objective was to compare the microbiota in two groups of patients with COPD of different severity in order to detect potential microbiological markers that could help to provide a better understanding of the pathogenesis of this disease and the role played by the microbiota in its severity. The current study recruited nine patients with mild or moderate COPD and 10 patients with severe or very severe COPD in a stable condition (at least 3 months without exacerbation or use of antibiotics for any other reason). Diagnosis and classification of COPD was established according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) recommendations [2]. After DNA extraction from good quality expectorated sputum, quantitative (q)PCR (7500 real time PCR system thermocycler; Life Technologies, Grand Island, NY, USA) was used to quantify the copy number of the 16S rRNA gene in each sample (total bacterial load), yielding a 468 bp amplicon located between nucleotides 340–808 in reference to the 16S rRNA gene of Escherichia coli strain MG1655 (National Center for Biotechnology Information (NCBI) reference sequence: NR_102804.1). In order to compare the results obtained, they were normalised to the number of human cells in the sample, quantified by qPCR, using the human albumin gene [3]. A region measuring 525 bp, between position 8 and 533 of the 16S rRNA gene was amplified and pyrosequenced (Roche GS FLX Titanium with Lib-L type microspheres; 454 Life Sciences, Branford, CT, USA). This region comprises the regions of gene hypervariability from V1 to V3 of the 16S rRNA gene …