Microbiota as the missing link in development of COPD? A 16S rRNA- based analysis of the microbiome in subjects with and without COPD.
A 16S rRNA-based analysis of the microbiome in subjects with and without COPD.
The project is part of the Bergen COPD Microbiome Study (MicroCOPD), which aims to compare the community of microorganisms (microbiome) in the airways of subjects with and without COPD.
The recent development of high throughput sequencing technologies has made possible the characterization of bacterial communities present in the lungs through sequencing of shorter regions of the bacterial 16S rRNA gene. This gene is unique in that it consists of conserved as well as hypervariable regions (V1-V9), that enables identification through large genomic libraries. However, the lack of standards in the field with regards to sequencing methodology and bioinformatic analysis makes comparisons across studies challenging. One of the aims of the current project is to show that microbial community descriptions generated when sequencing the 16S rRNA gene V4 region, which appears to be increasingly used as the standard, will be the same as when sequencing the 16S rRNA gene V3V4 region. This is of particular importance to the MicroCOPD study, which follows one of the commercial protocols (Illumina) for sequencing of the V3V4 region. Samples from 16 subjects with COPD and 7 healthy controls are included in the current project. The sample types per subject include protected specimen brushes and bronchoalveolar lavage collected by bronchoscopy, an oral wash sample and a negative control PBS sample. In addition a sample of known bacterial composition, referred to as a mock community is included on all sequencing runs. When comparing the microbial community descriptions generated from the sequencing of the different gene regions (V3V4 vs V4), we need to know that the variation we find is a result of true sequence variation and not differences in incorporated PCR and sequencing error. Data analysis will therefore involve the calculation of error rates based on the sequencing of the mock community sample for which the actual DNA sequences are known. The two main bioinformatic pipelines available for analyzing 16S amplicon data are QIIME (qiime.org) and mothur (www.mothur.org). The two pipelines differ in their recommended approaches to quality filtering and error correction. In the current study, the calculation of error rates will therefore also be of importance when evaluating the effect choice of bioinformatic pipeline has on final microbial community descriptions. As of January 2017, the sequencing of the V3V4 region of the 23 subjects is completed. The sequencing of the V4 region using the same samples and protocol is ongoing. Further, the sequencing of the V4 region using an alternative protocol with fewer rounds of PCR will be carried out. In parallell with this, the main projects carry on with sequencing of 126 individuals with COPD, 102 control subjects and 21 individuals with asthma. In addition to the work referred to above, three other papers are in the pipeline, of which one will be submitted within the first week of February 2017.