This is a central repository for 16S rRNA gene amplicon sequencing data generated from samples of biomass from the Samail Ophiolite, Oman. The purpose of this repository is to provide a fully documented, reproducible and update-able workflow for processing these data.
Most of the data processing was performed in R using the dada2 package. Data processing and outputs can be visualized as R Markdown-generated html files provided in the docs/rmarkdown folder. In addition to the standard R outputs, system commands to run non-R-based programs such as idemp for demultiplexing, cutadapt for demultiplexing and primer trimming, and Figaro for optimizing quality trimming parameters were run with the R system2() function, which allowed the system output to be saved and visualized through the R Markdown-generated html files. This aspect is an advantage over many DNA sequence processing pipelines, since system command outputs are often not saved or are hard to visualize and share.
This pipeline starts from the raw (usually multiplexed) fastq files provided by the sequencing facility and ends at sequence table and taxa table outputs from dada2. The raw fastq files are large (>1 Gb), and so they are not on Github. Instead, for data processing, they are saved on a local drive, with the paths to the files stored in scripts/paths.R. The raw data for this project can be downloaded from this Google Drive Folder, which also contains some related files such as demultiplexed fastqs, mapping files, and Figaro output, for convenience. To reprocess these data, the raw fastq files can be downloaded and their local paths set by editing paths.R.
There should not be much need to fully reprocess the data from the raw fastq files, since demultiplexing and quality filtering methods will probably not change drastically over the next few years. On the other hand, reference databases (such as Silva) for taxonomic assignments are updated regularly. This is one of the reasons the data processing steps have been separated into three files: OMXX_seq_prep.Rmd for demultiplexing, OMXX_dada2_ASV_table_generation.Rmd for trimming, quality filtering, and sample inference, and OMXX_taxa_assignment.Rmd for taxonomic assignment. The most up-to-date reference database at the time of writing is Silva 138, but when a newer database is released, it could be downloaded (formatted for dada2) and its local path set in paths.R. Then, taxonomic assignments could be readily updated by running/knitting the OMXX_taxa_assignment.Rmd files.
Samail Ophiolite 16S rRNA gene data are the products of the following works:
Miller, H. M., Matter, J. M., Kelemen, P., Ellison, E. T., Conrad, M. E., Fierer, N., et al. (2016). Modern water/rock reactions in Oman hyperalkaline peridotite aquifers and implications for microbial habitability. Geochim. Cosmochim. Acta 179, 217 – 241. doi:10.1016/j.gca.2016.01.033
Rempfert, K. R., Miller, H. M., Bompard, N., Nothaft, D., Matter, J. M., Kelemen, P., et al. (2017). Geological and geochemical controls on subsurface microbial life in the Samail Ophiolite, Oman. Front. Microb. 8, 1–21. doi:10.3389/fmicb.2017.00056
Kraus, E.A., Nothaft, D., Stamps, B.W., Rempfert, K.R., Ellison, E.T., Matter, J.M., Templeton, A.S., Boyd, E.S., Spear, J.R.. Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman. Appl Environ Microbiol 2021;87(2). URL: https://aem.asm.org/content/87/2/e02068-20. doi:10.1128/AEM.02068-20. arXiv:https://aem.asm.org/content/87/2/e02068-20.full.pdf.
Nothaft, D.B., Templeton, A.S., Rhim, J.H., Wang, D.T., Labidi, J., Miller, H.M., Boyd, E.S., Matter, J.M., Ono, S., Young, E.D., Kopf, S.H., Kelemen, P.B., Conrad, M.E., The Oman Drilling Project Science Team, . Geochemical, biological and clumped isotopologue evidence for substantial microbial methane production under carbon limitation in serpentinites of the Samail Ophiolite, Oman. Earth and Space Sci Open Archive 2020;URL: https://doi.org/10.1002/essoar.10504124.1. doi:10.1002/essoar.10504124.1.
Nothaft, D., Templeton, A.S., Boyd, E., Matter, J., Stute, M., Paukert Vankeuren, A.N.. Aqueous geochemical and microbial variation across discrete depth intervals in a peridotite aquifer assessed using a packer system in the samail ophiolite, oman. Earth and Space Science Open Archive 2021;:34URL: https://doi.org/10.1002/essoar.10506402.2. doi:10.1002/essoar.10506402.2.