Computational protocol: Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters

Similar protocols

Protocol publication

[…] A total of eight samples, including four each before and after fermentation, were collected, respectively. Sample and sequence information are tabulated (). DNA was extracted using the OMEGA DNA isolation kit (Omega, D5625-01, USA) following the manufacturer’s instructions. The quality of extracted DNA was checked by 1% agarose gel electrophoresis and spectrophotometry (optical density at 260 nm/280 nm ratio). All extracted DNA samples were stored at −20 °C for further analysis.The bacterial 16S rRNA was amplified by PCR for barcoded SMRT sequencing with the forward 27F (5′-GAGAGTTTGATCCTGGCTCAG-3′) and the reverse 1541R (5′- AAGGAGGTGATCCAGCCGCA-3′) primers. These primers contained a set of 16-nucleotide barcodes. PCR amplifications of the 16S rRNA regions were performed as described previously. The PCR program was as follows: 95 °C for 2 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s with a final extension of 72 °C for 5 min. The amplicons were sequenced using P6-C4 chemistry on a PacBio RS II instrument (Pacific Biosciences). The quality control for PCR amplifications and sequence preprocessing was performed as described previously.Raw data processing was carried out using the protocol RS_ReadsOfinsert.1 available in SMRT Portal version 2.7 as described previously. The extraction of high-quality sequences was firstly performed with the Quantitative Insights Into Microbial Ecology (QIIME) package (version 1.7). Then, PyNAST and UCLUST were applied to align the extracted high-quality sequences under 100% clustering of sequence identity to obtain representative sequences. The unique sequence set was classified into operational taxonomic units (OTUs) under the threshold of 98.6% identity using UCLUST after the selection of the representative sequences. The taxonomy of each OTU representative sequence was assigned using the Ribosomal Database Project (RDP) II database that classified at a minimum bootstrap threshold of 80%. A de novo taxonomic tree was constructed employing a representative OTU set in FastTree for downstream analysis, including the beta diversity calculation. The Shannon-Wiener, Simpson’s diversity, Chao1 and rarefaction estimators were calculated to evaluate the alpha diversity. The UniFrac distance was calculated based on the phylogenetic tree. Both weighted and unweighted calculations were performed for the principal coordinate analysis (PCoA). The graph presentations were generated by the R package version 3.1.2 and the Origin software version 8.5. The sequence data reported in this study have been deposited in the MG-RAST database (accession No. 4678995.3–4679002.3). […]

Pipeline specifications

Software tools QIIME, PyNAST, UCLUST, FastTree, UniFrac
Databases MG-RAST
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Bacillus megaterium, Pediococcus acidilactici, Lactobacillus plantarum
Diseases Alzheimer Disease
Chemicals Acetic Acid, Lactic Acid, Butyric Acid