Metagenomic, metatranscriptomics and 16S rRNA gene sequence data from diel sampling at Groves Creek Marsh, Skidaway Island, GA during July 2014 (NCEI Accession 0278832)

Acquisition Description:
Sample collection
Surface water samples were collected from approximately 1 m depth using a hand deployed Niskin bottle. Samples for dissolved constituents (dissolved organic carbon, colored dissolved organic matter, and nutrient analyses) were filtered on site through 0.2 m Polycap filters within minutes of collection and then transported to the laboratory for further processing. For cell counts by flow cytometry, samples were transported to the laboratory and fixed using 25% glutaraldehyde. For additional microbial analyses (microbial biomass collection for DNA and RNA extractions and bacterial production) were returned to the laboratory, which was within 10 minutes’ drive of the field site. Salinity was measured for discrete samples collected in the field using a handheld multiparameter probe (YSI, Pro2030). Depth was recorded using a YSI 600OMS V2 Optical Monitoring Sonde deployed on the creek bed.

Microbial community analysis sample collection
Planktonic microbial cells from surface water samples were collected by filtration. Water was pre-filtered through a GF/D glass fiber filter (~2.7 μM pore size, Whatman, GE Healthcare Life Sciences, Marlborough, MA); 500 mL of the filtrate was passed through a 0.22 μM pore size, 47 mm diameter filter (Millipore, Burlington, MA). Filtration was completed within 30 min of sample collection. After filtration all filters were placed in cryovials and flash frozen in liquid nitrogen. The samples were stored at -80 C until processing.

Microbial community analysis sample processing
Nucleic acids were extracted from samples following standard methodology. Briefly, for DNA the filters were thawed and placed in a 2 mL tube with 0.3 g glass and zirconia beads (0.2 g glass and 0.1 g zirconia), 0.75 mL CTAB extraction buffer, 0.75 mL phenol:chloroform:isoamyl alcohol (25:24:1, pH 8), internal standards, proteinase K, 10% SDS, and lysozyme for DNA extractions. Samples were vortexed for 10 min to lyse the cells. For RNA extraction, sample tubes were centrifuged for 10 min at 10,000 rpm and 4° C. The lysates were transferred to a sterile 1.5 mL microcentrifuge tube and mixed with 0.75 mL chloroform:isoamyl alcohol (24:1). The aqueous phase was added to a sterile 1.5 mL microcentrifuge with MgCl2, sodium acetate, and isopropanol. This solution was incubated at -80° C for 1.5 hours and then centrifuged at 4° C for 45 min at 10,000 rpm. The supernatant was discarded, and the RNA was washed with 70% EtOH twice. Following RNA extraction Turbo DNase was used to remove residual DNA. For metagenomic samples the lysate was centrifuged at 5,000 rpm for 5 min and washed twice with 0.5 mL of chloroform:isoamyl alcohol by centrifugation at 15,000 rpm for 5 min. The upper aqueous phase was incubated with isopropanol at room temperature for 2 hrs. The DNA was precipitated by centrifugation at 10,000 rpm for an hour and washed with 70% EtOH twice.

All sequencing, assembly, and annotation was performed by the DOE Joint Genome Institute (JGI). JGI generated 16S rRNA libraries, metagenomes, and metatranscriptomes. Plate-based DNA library preparation for Illumina sequencing was performed on the PerkinElmer Sciclone NGS robotic liquid handling system using Kapa Biosystems library preparation kit. DNA was sheared to 300 base pairs (bp) using the Covaris LE220 focused-ultrasonicator and size selected using SPRI beads (Beckman Coulter). The fragments were treated with end-repair, A-tailing, and ligation of Illumina compatible adapters (IDT, Inc) containing a unique molecular index barcode for each sample library. qPCR was used to determine the concentration of the libraries and were sequenced on the Illumina HiSeq-2500 to yield 150 bp paired-end reads at the DOE Joint Genome Institute. Quality filtered metagenomic sequences for each sample were assembled with metaSPAdes (version 3.10.1; and all contigs >200 bp were uploaded and annotated by the Integrated Microbial Genomes (IMG) pipeline. For metatranscriptomes, a plate-based RNA sample preparation was performed on the PerkinElmer Sciclone NGS robotic liquid handling system using the Illumina Ribo-Zero rRNA Removal Kit (bacteria) and the TruSeq Stranded Total RNA HT sample prep kit following the protocol outlined by Illumina. Total RNA starting material consisted of 100 ng per sample and included 10 cycles of PCR for library amplification. Illumina sequencing was performed as described for metagenome samples.

Quality filtered metatranscriptomic sequences for each sample were assembled with Megahit (version 1.10.6), and all contigs > 200 bp were annotated as described for the metagenome samples. Datasets which had assemblies for which the N50 was greater than three standard deviations from mean were not included in further analyses (Supplemental Tables 1 and 2) Resultant assemblies were combined with coding sequences (CDS) using bedtools2 (version 2.27.0) in order to generate an assembly with CDS embedded. Quality controlled raw reads were mapped to the assembly with gene features using bowtie2 (version 2.2.9). Coverage information on the number of reads mapping to each contig was generated using pileup in the BBmap suite of tools. The coverage information was used to normalize read counts to account for the length of reads and the length of CDS. Read counts within KEGG ortholog groups (KO) were summed and normalized as read counts per million mapped to KO-annotated contigs (genes per million [GPM], transcripts per million [TPM]). GPM and TPM were also used in taxonomic analyses.