Mobio powersoil dna extraction kit

Approximately, 200 µl of biofilm was extracted using MoBio PowerSoil DNA extraction kit (MoBio Laboratories, Inc., CA, USA) according to the manufacturer’s protocol, with the bead-beating time reduced to less than 1 min. This DNA extract was then gel purified and quantified using a low-mass ladder (Promega).
Total genomic DNA for metagenomic sequencing (150-bp or 250-bp reads) for both biofilm and planktonic samples (20% of each filter) was extracted using MoBio PowerMax Soil DNA extraction kit. Cells were extracted from 20% of each filter by adding 15 ml of lysis buffer and vortexing for 10 min. Lysis of cells was modified by heating to 65 °C for 30 min and 1 min of bead beating. DNA was eluted in Milli-Q water, and ethanol precipitation was performed (70% EtOH, 3-M sodium acetate, incubation for 24 h at 4 °C).

For each mosquito that blood-fed DNA was individually extracted from the dissected head, abdomen and legs of each insect using the MoBio Powersoil DNA extraction kit following manufacturer’s instructions (MoBio Laboratories Inc., Carlsbad CA USA). The insects were held by the wings and each body portion (legs, abdomen, head) individually and separately removed with sterile, fine forceps to avoid cross contamination during dissections. Body parts were stored individually (legs were pooled per insect) in sterile 1.5mL tubes at -20°C until DNA extraction. DNA was similarly extracted from mouse tissue. Procedural extraction control blanks (sterile water) were included at a frequency of 10% to monitor potential PCR contamination, in addition to no-template negative controls. IS2404 quantitative PCR (qPCR) was performed as described [33 (link)]. IS2404 cycle threshold (Ct) values were converted to genome equivalents (GE) to estimate bacterial load within a sample by reference to a standard curve (r² = 0.9312, y = [-3.000Ln(x)+39.33]*Z, where y = Ct and x = amount of DNA [fg] and Z = the dilution factor]), calculated using dilutions of genomic DNA from M. ulcerans strain JKD8049, quantified using a fluorimeter (Qubit, Invitrogen) [33 (link)].

The soil DNA was isolated by using MoBio PowerSoil DNA extraction kit (MoBio Laboratories, Carlsbad, CA, USA) following the manufacturer's instructions with a few alterations. Briefly, 0.5 g subsampled soil was mixed with the beads and lysing buffer. An additional 10 min incubation time was conducted at 65 °C, then the bead beating was performed for 2 min^{25 (link)}. A 0.8% (wt/vol) low melting point agarose gel was used to evaluate the DNA integrity. DNA was purified by using the agarose gel DNA purification kit (TaKaRa Bio USA, Inc., Mountain View, CA, USA). A NanoDrop One spectrophotometer was used to measure the DNA quantity (NanoDrop Technologies, Wilmington, DE, USA).

DNA was extracted from 0.5 g soil using a MoBio Power Soil DNA extraction kit (MoBio Laboratories, Carlsbad, CA, United States) by following the manufacturer’s instructions, purified with an Ultra Clean 15 DNA purification kit (MO BIO), and stored at −20°C. Bacterial community analysis was carried out using a 16s rRNA genes primer pairs 515F (5′-GTGCCAGCMGC CGCGGTAA-3′)/907R (5′-CCGTCAATTCCTTTGAGT TT-3′) for the V4 hypervariable regions (Biddle et al., 2008 (link)), the fungal ITS2 region was amplified by primer sets ITS3 (5′-GCATCGATGAAGAACGCAGC-3′)/ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (Gade et al., 2013 (link)). DNA concentration was measured on a Nano-Drop ND-1000 spectrophotometer (Thermo Scientific, United States). After sequencing, sequences were analyzed using the QIIME pipeline² (Caporaso et al., 2010 (link)). The low-quality sequences that had a quality score < 20, contained ambiguous nucleotides, or did not match the primer and barcode, were removed. Operational taxonomic units (OTUs) were generated at 97% similarity cutoff using the UCLUST method in QIIME (Edgar, 2010 (link)). Using Greengenes database³ to annotate taxonomic information for each bacterial sequence, UNITE database (Kõljalg et al., 2005 (link)) to identify fungal taxonomy.

We extracted microbial DNA from feces and biopsy (mucosal) samples using MOBIO PowerSoil DNA extraction kit (MOBIO Laboratories, Carlsbald, CA, USA). We prepared sequencing libraries using the protocols from Earth Microbiome project using V4 primers with Illumina Miseq Instrument^{62 (link)}. PANDAseq^{63 (link)} paired reads were analyzed using QIIME 1.9 suite^{34 (link)}. The details of the analysis can be found in the Supplementary Document. Briefly, OTUs were formed at 99% sequence similarity and the OTUs that contained less than 0.005% of the total number of sequences and chimeric sequences were omitted from the analysis as previously recommended⁶⁴ . We calculated alpha and beta diversity metrics of Phylogenetic Diversity Whole Tree^{65 (link)}, and Unifrac^{33 (link)}. Gene abundances for bile acid biosynthesis were predicted with Phylogenetic Investigation of Communities by Reconstruction of Unobserved Species (PICRUSt) software^{53 (link)}. Genus-level phylotypes that significantly differed after RYGB were clustered based on Euclidean distances using ClustVis^{66 (link)}.

The MoBio Power Soil DNA extraction kit (MoBio Laboratories, Carlsbad, CA, USA) was used to extract the DNA from samples collected along the ML chronosequence. The extracted DNA samples were sent to Macrogen Incorporated (Seoul, Korea) for sequencing. The V3 and V4 region of the 16S rRNA gene was amplified using bacterial primers Bakt_341F and Bakt_805R [35 (link)]. The resulting amplicons were sequenced using 300-bp pair-end Illumina MiSeq system (Illumina, San Diego, CA, USA). The 16S rRNA gene sequence data from the ML chronosequence was deposited in the MG-RAST server under project ID mgp21131 (http://metagenomics.anl.gov/linkin.cgi?project=mgp21131).