Sample preparation and 16S V4 sequencing analysis
Sponges were removed from their plastic stems and individually placed in 0.5-ml tubes (Eppendorf, Hauppauge, NY, USA), which had the distal end previously pierced using a sterile 18-gauge needle (BD Biosciences, San Jose, CA, USA); each of these individual tubes were then placed into 2-ml tubes (Eppendorf, Hauppauge, NY, USA). Bacteria were quickly eluted and pelleted by adding 100 μl of 25 mM HEPES, 50 mM NaCl, 1% Triton-X, 1 mM DTT, and 5 mM EDTA and centrifuging in an Eppendorf 5415D centrifuge (Eppendorf, Hauppauge, NY, USA) for 30 s. This collection step was repeated with another 100 μl of the elution buffer above. Supernatant was immediately removed and pellets were frozen in a -80° freezer (Model: ELT1786-9-D40, Thermo Scientific, Asheville, NC, USA) with a backup phone system, until further processing.
Genomic DNA was extracted from the 60 samples using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA), and a 30-second beat-beating step using a Mini-Beadbeater-16 (BioSpec Products, Bartlesville, OK, USA). High-throughput sequencing analysis of bacterial rRNA genes was performed using extracted genomic DNA as the templates. One hundred microliter amplification reactions were performed in an MJ Research PTC-200 thermal cycler (Bio-Rad Inc., Hercules, CA, USA) and contained: 50 mM Tris (pH 8.3), 500 μg/ml bovine serum albumin (BSA), 2.5 mM MgCl2, 250 μM of each deoxynucleotide triphosphate (dNTP), 400 nM of each primer, 4 μl of DNA template, and 2.5 units JumpStart Taq DNA polymerase (Sigma-Aldrich, St Louis, MO, USA). The PCR primers (F515/R806) targeted a portion of the 16S rRNA gene containing the hypervariable V4 region, with the reverse primers including a 12-bp barcode . All primer sequences are available in Additional file 1. Thermal cycling parameters were 94°C for 5 minutes; 35 cycles of 94°C for 20 seconds, 50°C for 20 seconds, and 72°C for 30 seconds, followed by 72°C for 5 minutes. PCR products were purified using a MinElute 96 UF PCR Purification Kit (Qiagen, Valencia, CA, USA). DNA sequencing was performed using an Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA, USA). Clusters were created using template concentrations of 1.9 pM and PhiX at 65 K/mm2, (manufacturer’s recommendations for samples with uneven distributions of A, C, G and T). One hundred base sequencing reads of the 5′ end of the amplicons and seven base barcode reads were obtained using the sequencing primers listed in Additional file 1. De-multiplexing, quality control, and operational taxonomic unit (OTU) binning were performed using Quantitative Insights into Microbial Ecology (QIIME) .
The total initial number of sequencing reads was 71,581,480. Low quality sequences were removed using the following parameters: Q20, minimum number of consecutive high-quality base calls = 100 bp, maximum number of N characters allowed = 1, maximum number of consecutive low-quality base calls allowed before truncating a read = 3. Numbers of sequences removed using the aforementioned quality control parameters were: barcode not in mapping file (35,296,547), reads too short after quality truncation (4,926,462), and too many Ns (5,431). Remaining reads numbered 31,353,040, which were then used to pick OTUs from the GreenGenes reference database (May 18, 2012 database); this database automatically bins OTUs at 97% identity, ensuring the resulting data were compatible with phylotypic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis. Due to alignment failure, an additional 1,511,116 reads were discarded during OTU picking, providing 29,841,924 reads for downstream analysis.