Hotstartaq master mix

DNA extraction was achieved using the DNeasy Blood and Tissue Kit (Qiagen). Primers for amplification and pyrosequencing analysis of the methylation status of MIR17HG were designed using proprietary pyrosequencing Assay Design Software v2.0 (Qiagen). Primer sequences are in Table 2. These primers were used to analyse all 10 CpG’s between chr13:91348952 and chr13:91348988. Assay designs were saved as.xml files and imported to Pyro Q-CpG Software v1.0.9, which generated a nucleotide dispensation order according to the manufacturer’s standard parameters. Suitable bisulphite treatment controls were chosen from the options provided by the software. PCR reactions contained 12.5μl of Qiagen HotStarTaq Master Mix (Qiagen), additional magnesium chloride to achieve a final concentration of 2mM, 200nM each of forward and reverse primers, 2ul of DNA eluted from the bisulphite conversion and sufficient water to make a final volume of 25μl. Thermal cycling conditions were 94°C for 12 minutes to activate the Taq polymerase followed by 40 cycles of 94°C for 10 seconds, 55°C for 20 seconds and 72°C for 30 seconds. PCR products were sequenced by pyrosequencing on a PyroMark ID system (Qiagen) following the manufacturer’s protocols. Percentage methylation at each of the four CpG sites was calculated by the Pyro Q-CpG Software. Pyrograms and analysis reports were exported from this software.

The presence of sulfonamide resistance genes sul1, sul2 and sul3 and the most commonly found trimethoprim resistance genes dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17 was investigated in the SMX/TRI-resistant bacteria by PCR as described [34 (link),38 (link)] using either QIAGEN HotStarTaq Mastermix (QIAGEN GmbH, Hilden, Germany) (sul1 and sul2), KAPA HiFi HotStart PCR Kit (KAPA Biosystems, Boston, USA) (sul3), Dream Taq Green PCR Mastermix (2x) (ThermoFisher Scientific, Waltham, USA) (dfrA1), or Kappa 2G Robust PCR kit (KAPA Biosystems, Boston Massachusetts, USA) (dfrA5, dfrA7, dfrA12 and dfrA17). The presence of integrase genes intI1, intI2 and intI3 was queried for by PCR and the variable region of class 1 integron characterised by PCR and Sanger sequencing as described elsewhere [39 (link)]. The diversity of promoters associated with detected class 1 integrons were characterised by Sanger sequencing as already described [40 (link),41 (link)]. The sequenced sections of the class I integrons were deposited at GenBank under accession numbers MK093863 –MK093896.

Samples that were malaria-positive by either the pfldh qPCR or the All Plasmodium qPCR were tested using species-specific nested PCR as in [13 (link)]. The Round 1 reaction used the same primers as used in the genus-specific nested PCR but with Qiagen HotStarTaq Master Mix (Qiagen, Hilden, Germany) (Additional file 1: Table S1).
Separate Round 2 reactions were run for P. falciparum (primers rFAL1 and rFAL2), P. malariae (rMAL1 and rMAL2), and P. ovale (rOVA1 and rOVA2) (see Additional file 1: Table S1). The PCR products from each speciation reaction were analysed by gel electrophoresis on a 3 % agarose gel to determine positivity for each species in each sample. Samples that did not amplify for any of the three species were labelled ‘indeterminate’ by this method.
The species of each sample was determined based on results from nested PCR. If a sample was indeterminate by nested PCR, the BLAST result was used instead. If the BLAST result was also indeterminate, the sample was removed from the analysis.

Frozen aliquots of the fecal samples of the 25 donors were analyzed by Novogene (Beijing, China). DNA were extracted with the QIAamp DNA stool mini kit (Qiagen, Valencia, CA, USA) following the protocol provided by the supplier. Extracted genomic DNA (2 ng/μL) was used for library preparation. The purity and integrity of the DNA was determined with a nanodrop (ND-1000) spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) through 1% agarose gel electrophoresis (AGE). DNA concentration was measured using Qubit^® dsDNA Assay Kit in Qubit^® 2.0 Fluorometer (Carlsbad, CA, USA). The OD value was between 1.8–2.0, and DNA contents above 1μg were used to construct the library. DNA samples were amplified using 16S V3−V4 primers. Specific forward and reverse primers (F-NXT-Bakt-341F: 5′-CCTACGGGNGGCWGCAG-3′ andR-NXT-Bakt-805R: 5′-GACTACHVGGGTATCTAATCC-3′) were used in the PCR reactions procedure. Amplifications were performed in 25 µL reactions with Qiagen HotStar Taq master mix (Qiagen Inc, Valencia, CA, USA), 2 µL of primers, and 1 µL of template. Raw sequence data from the Illumina NovaSeq 6000 (Illumina^® Inc. Columbia Circle, Albany, NY, USA) platform was processed, and 250 bp paired-end reads were generated. Microbiota identification was carried out by clustering the operational taxonomic units (OTU) from phylum to genus level using MUSCLE (Version 3.8.31) software.

Tail snip DNA was extracted using KAPA Express Extract Kit (Roche). Genotyping PCR was performed with HotStarTaq Master Mix (Qiagen) according to manufacturer’s instruction. Genotyping primers used are summarized in Table S3. To determine the zygosity of HSC-Scl-CreER^T, qPCR was additionally performed with purified tail snip DNA using SYBR^™ Green Universal Master Mix (Applied Biosystems).