Iproof dna polymerase

To generate endogenously tagged small gatekeeper kinases, the putative kinase-coding genes were amplified from genomic DNA by PCR and cloned into the pKS_3HA_Neo vector [29 (link)]. Primer sequences and restriction enzymes are shown in S1 Table. PCR amplifications were performed using iProof DNA polymerase (Bio-Rad). Typically, an amplicon of ~ I kb in length that lacked the start codon was cloned in frame to a C-terminal triple-hemagglutinin epitope tag (3xHA) into the pKS_3HA_Neo plasmid. The plasmids were linearized by the enzyme reported in S1 Table and ~5 μg of DNA was used to transform wild-type Giardia. Transformants were selected with G418 at 40 μg/mL.
For protein expression, the complete coding region of each protein kinase was PCR amplified from Giardia genomic DNA. PCR amplicons were cloned into the ligation independent cloning (LIC) site of (MBP)-AVA0421 expression vector and validated by sequencing [30 (link)]. Recombinant proteins were expressed in E. coli BL21 (DE3), Invitrogen, Carlsbad, CA) using Studier auto-induction protocols at 20°C [31 (link)]. Recombinant protein purifications were performed as previously described [26 (link)].

DNA was extracted from 100–200 mg of stool using the E.Z.N.ATM Stool kit (Omega Bio-Tek Inc, GA, USA) according to the manufacturer’s protocol. Mechanical disruption of cells was carried out with the MP Bio FastPrep-24 for five cycles of 1 min at 5.5 M/s. 16S variable region 4 (V4) amplifications were carried out using the KAPA2G Robust HotStart ReadyMix (KAPA Biosystems) and barcoded primers 515 F (GTGCCAGCMGCCGCGGTAA) and 806 R (GGACTACHVGGGTWTCTAAT)^{55 (link)}. The cycling conditions were 95 °C for 3 min, 22 cycles of 95 °C for 15 s, 50 °C for 15 s and 72 °C for 15 s, followed by a 5 min 72 °C extension. Libraries were purified using Ampure XP beads and sequenced using MiSeq V2 (150 bp × 2) chemistry (Illumina, San Diego, CA). 18S V4 + V5 amplification was achieved using the iProof DNA polymerase (Bio-Rad Laboratories, Hercules, CA) with primers V4-1 (GCGGTAATTCCAGCTC) and V4-4 (GCCMTTCCGTCAATTCC) as previously described^{13 (link)}. Briefly, the cycling conditions used were 94 °C for 3 min, 30 cycles of 94 °C for 45 s, 56 °C for 1 min, and 72 °C for 1 min, followed by a 10 min 72 °C extension. Barcodes were ligated, and libraries were sequenced using MiSeq V3 (300 bp × 2) chemistry (Illumina, San Diego, CA). Sequencing was performed at the Centre for the Analysis of Genome Evolution and Function (Toronto, Canada).

Genomic DNA was extracted as described previously (Zolan and Pukkila, 1986 (link)). The coding region of the cag1 gene was amplified using four sets of primers (Table S1) with iProof DNA polymerase (Bio-Rad), subjected to agarose gel electrophoresis, purified from the agarose gels with GENECLAEN II Kit (Bio101), and used as templates for cycle sequencing reactions with BigDye Terminator v3.1 (Applied Biosystems). Sequencing was performed by the Biotechnology Center at Akita Prefectural University.

L, M, and S segments from qRT-PCR positive samples were amplified and sequenced using the primers in Table S1. Using Superscript III reverse transcriptase (ThermoFisher Scientific, Waltham, MA, USA) and IProof DNA Polymerase (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions, amplicons of each segment were made. The PCR products of the corresponding sizes were excised and purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA), then sent for Sanger sequencing at the RML Research Technologies Branch Genomic Core using the primers listed in Table S1. Phylogenetic analysis was done using Geneious Prime 11 2019.0.4 (Biomatters Ltd., San Diego, CA, USA). Analysis was performed using available sequences on GenBank matching the regions we sequenced. Reassortment analysis was done through manual comparison of the phylogenetic trees.

Kits for gel purification, plasmid-preparation, RNA-preparation (RNeasy), qRT-PCR, and RNA-Protect reagent were purchased from Qiagen. iProof DNA polymerase and deoxynucleoside triphosphates were purchased from Biorad.