Computational protocol: Microbial Community Response to Terrestrially Derived Dissolved Organic Matter in the Coastal Arctic

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Protocol publication

[…] Whole water from the sample sites and each incubation bottle was filtered onto a 0.2-μm Supor filter and frozen at -80 °C. Before extraction, 900 μL of DNA extraction buffer (), lysozyme (2 mg/mL final concentration) and proteinase K (0.2 mg/mL final concentration) were added to the filters. Samples were then subjected to three freeze-thaw cycles. This was followed by enzymatic lysis in a 30 min 37°C incubation and then continued lysis at 65°C for 1 – 2 h after the addition of SDS (1% final concentration). Two phenol:chloroform:isoamyl alcohol (25:24:1) extractions were then carried out to isolate nucleic acids. Nucleic acids were then precipitated using 100% isopropanol (0.6 × volume of the resulting supernatant) for 2 h up to overnight, pelleted at 13,000 rpm for 30 min, and then rinsed and re-pelleted twice with 70% ethanol before being dried in a roto-evaporator. Once dry, samples were resuspended in 250 mL of nuclease-free water.The V4 region (515F, GTGCCAGCMGCCGCGGTAA and 806R, GGACTACHVGGGTWTCTAAT) of the 16S rRNA gene for prokaryotic composition was amplified using Earth Microbiome Project protocols, but with only 30 total cycles. This 806R primer has a bias against SAR11 sequences () and thus the relative abundance of these taxa and Alphaproteobacteria in general are likely underestimated in our study. Sample libraries were sent to Argonne National Lab for 2 × 150 bp sequencing on the Illumina MiSeq platform and reads were paired using fastq-join (). Sequences that successfully joined were quality filtered, dereplicated (derep_fulllength) and abundance sorted (sortbysize) using UPARSE v 7.0.1001 (fastq_filter) () with an expected error rate of 0.5. Singleton sequences were removed in the latter step in order to prevent them from seeding clusters when clustering operational taxonomic units (OTU). Reads were then clustered (cluster_otus in UPARSE pipeline) at 97% similarity. Reference-based chimera filtering was performed using UPARSE (uchime) with the Gold Database as the reference database. Reads (including singletons) were subsequently mapped back to OTUs using UPARSE (usearch_global) and an OTU table created. Taxonomy of the representative sequences was assigned in QIIME v 1.8 (assign_taxonomy.py; ) using the RDP classifier trained to the Greengenes database (v. 13.8) for 16S amplicons. Any remaining singletons and OTUs occurring in only one sample, chloroplast, mitochondrial and archaeal sequences were removed in QIIME (filter_otus_from_otu_table.py). Sequences passing these quality control filters were uploaded to the NCBI Short Read Archive (SAMN06175504 to SAMN06175533) under the BioProject PRJNA310254. Samples were subsampled to 3037 sequences per sample (the minimum number of sequences per sample in the dataset) before subsequent analyses. […]

Pipeline specifications

Software tools ea-utils, UPARSE, UCHIME, USEARCH, QIIME, RDP Classifier
Databases Greengenes EMP
Application 16S rRNA-seq analysis
Diseases Tooth Erosion
Chemicals Carbon, Nitrogen