Data on Lung and Intestinal Microbiome in Ageing Rats exposed to Air Pollution
Description
This dataset reflects the lung and intestinal microbiome in Fischer 344 ageing rats exposed to traffic-related air pollution for 3 months. Data here include lung and intestinal microbiome identified by analysing the 16S ribosomal (r)RNA gene amplified with specific primers, including Illumina sequencing adapters and sample-specific barcodes, and sequenced on an Illumina MiSeq sequencer.
Files
Steps to reproduce
To extract DNA of lung bacteria of each rat, the left lobe of the lung was harvested under sterile conditions, and 10 mg fresh sample was used in QIAamp DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany). The QIAamp DNA Stool Mini Kit (Qiagen) were used to extract intestinal bacterial DNA following the manufacturers’ instructions. The minimum final concentration of intestinal and lung bacterial DNA samples was 5 ng/µL, and all DNA samples were stored at -80 °C. Universal 16S ribosomal (r)RNA gene primers V3 (341F, 5’-CCTACGGGNGGCWGCAG-3’) and V4 (805R, 5’-GACTACHVGGGTATCTAATCC-3’) were recommended and designed by Illumina (https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html). These two primers involved overhang adapter sequences in the forward (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3') and reverse (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3') primers and amplified the targeted sequence of the bacterial 16S rRNA gene. In addition, a limited cycle polymerase chain reaction (PCR) amplified the V3-V4 region of the bacterial 16S rRNA gene to construct the amplicon library. The sequencing libraries, Illumina sequencing adapters, and dual-index barcodes were attached to the amplicon library. To ensure that the amount was sufficient to attach 16S rRNA, the quantity and quality of the sequencing libraries were confirmed by a QSep100 analyzer (BiOptic, New Taipei City, Taiwan). The v3 chemistry generated paired-end reads of 300 bases in length to normalize the libraries, pool the library in an equimolar ratio, and sequence them on Illumina Miseq. After 16S rRNA sequencing, the universal primer sequence and low-quality reads were removed. The following process and analysis were executed with the phyloseq workflow of the DADA2 package (vers. 1.6) in R environment. Functions of the DADA2 package included filtering, trimming, de-replication, and de-noising of the forward and reverse reads. After merging the processed overlapping paired-end reads, chimers were removed from the cleaned full-length amplicons. Taxonomic assignment of the inferred amplicon sequence variants (ASVs) was performed using the SILVA reference database (vers. 132) with minimum bootstrap confidence of 80. Multiple sequences were aligned to ASVs with the DECIPHER package (vers. 2.6.0), and RAxML (vers. 8.2.11) was used to construct a phylogenetic tree. The phyloseq package (vers. 1.22.3) created a phyloseq object for downstream bacterial community analyses based on the frequency table, taxonomy, and phylogenetic tree information.