Taqpath proamp master mix

We genotyped three SNPs in the DDAH1 gene (rs1241321, rs480414, and rs233112), two SNPs in the DDAH2 gene (rs805304 and rs2272592), two SNPs in the AGXT2 gene (rs16899974 and rs37369), and two SNPs in the PRMT1 gene (rs1041588 and rs975484). These SNPs were selected on the basis of a literature search for SNPs that have been reported to be associated with differences in ADMA concentration, differences in nitric oxide-related vascular function, or cardiovascular diseases (Appendix A). Details of published information on the selected SNPs is given in Appendix A Table A1. SNP genotyping was performed using single-tube human TaqMan SNP Genotyping Assays (Thermofisher, Waltham, MA, USA). Each reaction mix contained 10 ng template DNA, 0.5 µL specific TaqMan SNP Genotyping Assay, and 5 µL of 2xTaqPath ProAmp™ Mastermix in a total reaction volume of 10 µL. The PCR was performed in a QuantStudio™ 5 Real-Time PCR System (Thermofisher, Waltham, MA, USA) using the following PCR program: pre-read of 30 s at 60 °C, enzyme activation at 95 °C for 5 min, 40 cycles of denaturation (5 s, 95 °C) and annealing (30 s, 60 °C), followed by a last post-read of 30 s at 60 °C. Allelic calls were identified by Quant Studio Design and Analysis Software (Thermofisher, Waltham, MA, USA).

The M224L polymorphism (rs2234970) of the SCD1 gene was genotyped by own-designed TaqMan assay. The assay contained the two primers (sense: 5′–CAC AAG CGT GGG CAG GAT–3′, antisense: 5′–GGT GTC TGG TCT GTC AAT GTA GGT–3′) and the allele-specific fluorescent probe labelled by JOE for the C allele (5′–JOE-AAG CAC ATC AGC AGC AAG CCA GGT T-BHQ1–3′) and labelled by FAM for the A allele (5′–FAM-AAG CAC ATC ATC AGC AAG CCA GG-BHQ1–3′). Real-time PCR assay was performed in 10 µL final volume containing approximately 4 ng genomic DNA, 1× TaqPath™ ProAmp™ Master Mix, 0.5 µM primers and 0.3 µM allele-specific probes using QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Thermocycle was started by activating the hot start DNA-polymerase and denaturing genomic DNA at 95 °C for 10 min. This was followed by 40 cycles of denaturation at 95 °C for 15 s, and combined annealing and extension at 60 °C for 1 min. Real-time detection was carried out during the latter step to verify the results of the subsequent post-PCR plate read and automatic genotype call.

Rs6841081 and rs3811792 polymorphisms of the SCD5 gene were genotyped using pre-designed TaqMan assays (C_34192814_10 and C_27029625_20, Thermo Fisher Scientific, Waltham, MA, USA). qPCR assay was performed in 5 µL final volume containing approximately 4 ng genomic DNA, 1× TaqPath™ ProAmp™ Master Mix, and 1× TaqMan^® SNP Genotyping Assay using QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Thermocycle was started by activating the hot start DNA-polymerase and denaturing genomic DNA at 95 °C for 10 min. This was followed by 40 cycles of denaturation at 95 °C for 15 sec, and combined annealing and extension at 60 °C for 1 min. Real-time detection was carried out during the latter step to verify the results of the subsequent post-PCR plate reads and automatic genotype calls.

DNA isolations were subjected to multi-parallel qPCR for the detection of G. duodenalis and for IAC analysis. A previously validated assay targeting the small subunit ribosomal RNA gene was used for the detection of G. duodenalis.³⁶ Stool-derived and swab-derived DNA extracts were run on separate 96-well plates, with each sample in duplicate 7 μL reactions. This study used TaqPath ProAmp Master Mix (ThermoFisher Scientific, Waltham, MA) and the Applied Biosystems™ StepOnePlus™ Real-Time PCR System (ThermoFisher Scientific, Waltham, MA). Primer oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Fluorescently labeled probes featured a 5′ FAM (fluorescein) fluorophore, an internal ZEN™ quencher, and a 3′ 3IABkFQ quencher (Integrated DNA Technologies, Inc., Coralville, IA). Cycling conditions entailed an initial 2-minute incubation at 50°C, followed by a 10-minute incubation at 95°C and 40 cycles of 1) a 15-second denaturation step at 95°C and 2) a 1-minute annealing and extension step at 60°C for the G. duodenalis assay and at 59°C for the IAC assay.

All DNA samples extracted at EPFL were subjected to quantitative PCR (qPCR) analysis to detect M. leprae prior to WGS. The repetitive element RLEP was quantified using TaqMan^® PCR amplification as described previously, with minor modifications (Truman et al., 2008 (link)). A total of 3 μl of each purified DNA sample, or the positive control (DNA from Thai-53, NR-19352) or the negative control (water), was added to a total PCR reaction volume of 20 μl containing 10 μl of TaqPath ProAmp master mix (Thermo Fisher Scientific, MA, United States), 900 nM of each forward (RLEPq-F) and reverse (RLEPq-R) primer, and 250 nM of the hydrolysis probe (RLEPq-P) (Table 1). The reaction mixtures were prepared in triplicate and amplification started with an initial denaturation step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min 60°C, using the QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, MA, United States). Data analysis was performed with the Thermo Fisher Connect Cloud², and the mean cycle threshold (C_t) was calculated for each sample. qPCR values were also used to evaluate the relative amount of M. leprae DNA in each sample and provide a GO/NO GO answer prior to WGS.

DNA was isolated from cryopreserved hepatocyte lots using the QIAamp DNA mini kit (Qiagen, Germany). Genotyping was performed using TaqMan SNP Genotyping Assays and TaqPath™ ProAmp™ Master Mix (Thermo Fisher Scientific, USA) as per manufacturer’s recommendations for polymorphisms in PNPLA3, MBOAT7, HSD17β13, and TM6SF2 (Table S2). PCR was performed and analyzed using the QuantStudio™ 5 Design and Analysis Software v1.5.1 (Applied Biosystems, USA).

Four hundred MK were lysed with water, RNA isolated and cDNA generated using Cells-to-Ct kit (Thermo Fisher), and assayed with Rnc2 Taqman Assay (Mm04260181_s1, Thermo Fisher Scientific, USA), mouse Tfrc Gene Copy Reference Assay (Thermo Fisher Scientific Cat# 4458367) and TaqPath ProAmp Master Mix (Thermo Fisher Scientific Cat# A30865) in an Applied Biosystems 7500 Real-Time PCR System. ddCt was calculated using Tfrc as control, Rnc2 is located within the unique mitochondrial DNA sequence^{88 (link)}, relative copy numbers of mtDNA were calculated using ddCT and ploidy of the cells: Relative mtDNA Copy Number = 2^-ddCt × ploidy/2. TaqMan Assays (Thermo Fisher) 4352933E (Actb, housekeeper), Mm00494336_m1 (Zfpm1/Fog-1), Mm00801891_m1 (Nfe2), Mm01352636_m1 (Gata1), Mm00492301_m1 (Gata2) and Mm00437040_m1 (Tpo) were also used. Applied Biosystems 7500 System Software v1.5.1 was used for real-time PCR data collection.