Powerbiofilm dna isolation kit

Metagenomic DNA was extracted from each sample core with the Power Biofilm DNA Isolation Kit (MO BIO Laboratories, Inc.), and pooled prior to sequencing. One round of extraction and pooling allowed obtaining enough DNA (pooled samples included DNA from three extractions, one from each triplicate core). Sequencing was performed at INDEAR genome sequencing facility (Argentina). TruSeq libraries were prepared according to TruSeq ® DNA Sample Preparation Guide Illumina (July 2012), starting from 2 μg DNA. Paired end libraries were sequenced with an Illumina HiSeq 1500 instrument. Raw sequence data have been deposited in the ENA European Nucleotide Archive (ENA) under the accession number ERP107533.

We collected pellets from BALF after centrifugation for 30 min at 13,000 g at 4 °C and the removal of supernatants. We stored pellets at −80 °C until processing. DNA extraction from the pellets was performed using a PowerBiofilm DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA).

The biofilm attached on a fiber was extracted directly from the fiber using a PowerBiofilm DNA Isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions; the DNA concentration was measured by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). The quantitative polymerase chain reaction (qPCR) using a CFX96 Touch real-time PCR Detection System (Bio-Rad, Herculers, CA, USA) with the fluorescent dye SYBR-Green approach was employed for amplifying the functional genes. The denitrification functional genes were periplasmic nitrate reductase (napA), membrane-bound nitrate reductase (narG), cytochrome cd1 nitrite reductase (nirS), copper nitrite reductase (nirK), nitric oxide reductase (cnorB), and nitrous oxide reductase (nosZ) genes. The functional genes for fixing inorganic carbon were for ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO): cbbL and cbbM. The primer sequences are provided in Table S1.
The qPCR amplification was performed in 20-μL reaction mixtures containing 10 μL of iTagTM Universal SYBR Green Supermix (Bio-Rad), 1 μL of template DNA (sample DNA or plasmid DNA for standard curves), 1 μL of forward and reverse primers respectively, and 7 μL of ddH₂O. The qPCR protocol involved 35–40 cycles. The results of qPCR were normalized by DNA concentration for the comparing of different DNA samples.

Microbial composition of prokaryotes and eukaryotes was assessed by DNA metabarcoding. Total genomic DNA was extracted using the Power Biofilm® DNAIsolation Kit (MoBio Laboratories, USA) following recommendations in Corcoll et al. (2017 (link)). DNA was precipitated with sodium acetate and ethanol prior downstream analyses. Bacterial 16S rRNA genes (V3 region) and eukaryotic 18S rRNA genes (V9 region) were amplified using V3 primers (modified primers 341F and 518R) (Klindworth et al., 2013 (link)) and V9 primers (1380F and 1510R) (Amaral-Zettler et al., 2009 (link)), respectively. Triplicate PCR reactions were performed for each sample to minimize potential PCR bias. The PCR amplicon libraries were sequenced using the Ion Torrent Proton technology according to the manufacturer's protocols.

Genomic DNA was extracted using a PowerBiofilm DNA Isolation kit (Mo Bio Laboratories Inc.) following the manufacturer’s protocol. The concentration and quality of DNA were measured with a Nanodrop spectrophotometer (Nanodrop 8000, Thermo Scientific). The extracted DNA (300 ng) was used as template for 16S rRNA gene fragments amplification and further pyrosequencing done at the Research and Testing Laboratory (Lubbock, TX, USA) using a Roche 454 FLX platform with the universal primers 28F (5′-GAGTTTGATCMTGGCTCAG-3′) and 519R (5′-GWATTACCG CGGCKGCTG-3′; Turner et al., 1999 (link)). Raw pyrosequencing reads have been deposited in the sequence read archive (SRA) of NCBI under accession number SRP068879.

Five samples (∼1 cm² each) from different segments of each lamina of the large conical mat were sliced with a sterile scalpel, transferred into separate microcentrifuge tubes consisting of beads (MO BIO Laboratories Inc., Carlsbad, CA, United States), and homogenized in a high velocity bead beater (BIO101/Savant FastPrep FP120 – Qiagen, Inc., Valencia, CA, United States). DNA was then purified using the PowerBiofilm^®DNA isolation Kit (MO BIO Laboratories) (Abbasian et al., 2015 (link)). The large conical mats used in this study are unique, found only in Lake Untersee. Therefore, to minimize sample collection while obtaining sufficient DNA to investigate the microbial composition of the mat laminae, we used five spatially representative samples from each lamina, and pooled the purified DNA into single samples for amplicon sequencing. The concentration and the quality of the pooled DNA from each mat lamina was determined by using a Lambda II spectrophotometer (Perkin Elmer, Norwalk, Conn.) followed by agarose gel electrophoresis (1% wt/vol agarose in 1X Tris-Acetate-EDTA (TAE) buffer, pH 7.8) to confirm that each sample consists of mostly high molecular weight DNA (>2 kbp) (Ausubel et al., 1987 ). The DNA was then dried in a Savant Speedvac Evaporator SVC 100H and stored at 4°C until used for sequencing.

Genomic DNA was extracted using PowerBiofilm DNA Isolation kit (Mo Bio Laboratories Inc.) following the manufacturer’s protocol. The concentration and quality of DNA were measured with a Nanodrop spectrophotometer (Nanodrop 8000, Thermo Scientific). Illumina Miseq sequencing was used with two different set of primers. First, total DNA from samples in 2013 and 2014 was used as a template for the V4 region amplification of the 16S SSU rRNA with the primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) (Caporaso et al., 2011 (link)), done at the Research and Testing Laboratory Genomics (Lubbock, Texas, United States). Second, samples from 2016 were used to amplify the V4-V5 region of the 16S SSU rRNA with the primers 515F-Y (GTGYCAGCMGCCGCGGTAA) and 926R (CCGYCAATTYMTTTRAGTTT) (Parada et al., 2016 (link)), done at LGC Genomics Gmbh (Berlin, Germany). The 515F-Y/926R primer improves the underestimation of SAR11 clade and the overestimation of Gammaproteobacteria produced by the 515F/806R primer (Parada et al., 2016 (link)). Raw amplicons reads were deposited in the sequence read archive (SRA) of NCBI under accession number SRP136789 and SRP136788.