Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4.

ABSTRACT:

Background

Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls.

Results

The number of sequences and amplicon se

SUBMITTER: Drengenes C 

PROVIDER: S-EPMC7784388 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature