Powerclean dna clean up kit

gDNA was extracted from ex vivo matured schizont-stage parasites (Qiagen
DNA blood midi kit, Germantown, USA). The gDNA was further purified with a PowerClean DNA
cleanup kit (Mo Bio Laboratories, Carlsbad, USA). Five micrograms of gDNA were
subsequently used for library preparation. SMRTbell DNA libraries (Pacific Biosciences,
Menlo Park, USA) were constructed according to the PacBio standard protocol with
BluePippin size selection (Sage Science, Beverly, USA), and sequences were generated on a
PacBio RSII instrument using P6-C4 chemistry.

SMRTbell libraries were constructed according to manufacturer’s instructions using the Greater Than 10 kb Template Preparation Using AMPure PB Beads and Sequencing (MagBead Station) protocols (Pacific Biosciences, Menlo Park, CA, USA). The extracted DNA was purified using a PowerClean DNA Clean-Up Kit (MoBio laboratories, Carlsbad, CA, USA). Small fragments of DNA were removed using AMPure PB Beads (Pacific Biosciences) of 0.45x. SMRTbell 20-kb libraries were prepared using a SMRTbell Template Prep Kit 1.0 (Pacific Biosciences). Libraries were subsequently sequenced on PacBio RS II (Pacific Biosciences) using DNA/Polymerase Binding Kit P6 v2 (Pacific Biosciences) and a DNA Sequencing Reagent Kit 4.0 (Pacific Biosciences). The titration density was 0.025 nM. The template was loaded into SMRT Cell v3 (Pacific Biosciences) using a Mag Bead Kit (Pacific Biosciences). Sequencing was performed using 30 cells in total. A 240-min movie was recorded for each cell. Metagenomic assembly was performed using a Hierarchical Genome Assembly Process (HGAP)^{27 (link)}. Minimus2 was used to attempt to overlap and join the resulting contigs^{28 (link)}. Final assemblies were achieved by polishing the draft assemblies using Quiver^{27 (link)}.

Whole-genome amplification from small quantities of DNA was performed using a REPLI-g Midi kit (Qiagen). Amplified DNA was purified using the PowerClean DNAClean-Up Kit (MO BIO Laboratories) and sample concentrations were calculated using the Qubit® dsDNA HS Assay Kit (Life Technologies). Library preparation was completed using the Nextera DNA Sample Preparation kit (Illumina) according to the manufacturer’s recommendations. Libraries concentrations were evaluated using the Qubit® dsDNA HS Assay Kit (Life Technologies) and the average library size was quantified using Experion (Bio-Rad). Libraries were then pooled in equimolar ratios and shotgun sequenced on the Illumina Miseq sequencing platform at MR DNA Research lab (Shallowater, TX) [34 (link)].

DNA concentrations were measured using Qubit^® dsDNA High Sensitivity Assay Kit (Life Technologies, Carlsbad, CA, USA). Whole genome amplification (WGA) was performed using REPLI-g Midi kit (Qiagen, Valencia, CA, USA). Amplified DNA was purified using PowerClean DNAClean-Up Kit (MO BIO Laboratories, Carlsbad, CA, USA) and quantified on Qubit^® dsDNA High Sensitivity Assay Kit (Life Technologies). Libraries were prepared with Nextera DNA Sample preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The concentration of the libraries was evaluated using Qubit^® dsDNA High Sensitivity Assay Kit (Life Technologies, Carlsbad, CA, USA) Then, libraries were pooled in equimolar amounts and shotgun sequenced on an Illumina MiSeq paired-end platform [19 (link)].

Total genomic DNA of each sediment sample was extracted using Powersoil DNA extraction kit (Mo bio Laboratories) according to the manufacturer's instructions. The crude DNA extracted was further purified using the PowerClean DNA Clean‐Up Kit (Mo Bio laboratories) for subsequent PCR‐DGGE analysis and Illumina MiSeq sequencing.

DNA extraction was performed using lysozyme (1 mg/ml) for 1 h at 37°C, as previously described [25 (link)]. Then proteinase K (0.2 mg/ml) and 1% sodium dodecyl sulfate (SDS) were added, and the samples were incubated at 55°C for 60 min with gentle agitation. The lysate was rinsed into a new tube with 1 ml SET buffer. Metagenomic DNA was extracted with one volume of phenol:chloroform:isoamyl alcohol (25:24:1) and then precipitated with ethanol and sodium acetate (0.3 M final concentration) at -20°C overnight. The DNA was purified using a Power Clean DNA Clean-Up Kit (MO BIO Laboratories) and stored at -20°C.