Qx200

For quantification of gene copy numbers, a ddPCR system was used (QX200, Bio-Rad Laboratories, Hercules, USA). DdPCR-reactions were prepared in 96-well plates with QX200 ddPCR EvaGreen Supermix (Bio-Rad) with a final volume of 22 µl and a primer concentration of 100 nm according to the manufacturer’s protocol. Plates were sealed by heat and for droplet generation an automated droplet generator (AutoDG Instrument, Bio-Rad) was used. A three-step PCR amplification was performed with a ramp rate of 2 °C/s under the following cycling conditions: 3 min at 95 °C, 40 cycles including 30 s at 95 °C, 1 min at primer specific annealing temperature (see Table 1) and 2 min at 72 °C, followed by 5 min at 4 °C and 5 min at 90 °C for signal stabilization of the PCR. The optimal annealing temperature of the newly designed primer pair was determined based on temperature gradient experiments (Fig. 1). Final quantification of Comammox amoA gene copy numbers in lake water samples was performed using ComaA1A2_336F/497R primer set (Table 1). Fluorescence signals were measured using a droplet reader (QX200, Bio-Rad) according to the protocol of the manufacturer, subsequent analyses of data were performed using QuantaSoft Analysis Pro Software (Bio-Rad) with a final visual check to evaluate the reliability of the automated threshold settings.

ddPCR analysis performed by the QX200 (BioRad) system combines water-oil emulsion droplet technology with microfluidics. Each sample is partitioned into 20,000 droplets by a droplet generator and each droplet is amplified by PCR. Then, droplets are analyzed by a droplet reader, which counts the positive and negative fluorescent droplets to define the target concentration. The NOTCH1^mut detection by ddPCR was conducted using the specific PrimePCR ddPCR Mutation Assays dHsaCP2500501 and dHsaCP2500500 (BioRad) according to the manufacturing protocol. ddPCR was performed by adding 5U of restriction enzyme HAE III (New England Biolabs) with 130ng of DNA template in a final volume of 20μl. After amplification, the 96-well PCR plate was loaded on the Bio-Rad QX200 droplet reader and ddPCR data were analyzed with QuantaSoft analysis software (version 1.7.4). The latter measures the number of positive and negative droplets for each probe (NOTCH1^mut and NOTCH1^wt) in each sample and calculates the fraction of positive droplets by a Poisson algorithm to determine the concentration of the target. Then, the software returns those data as the fractional abundance (FA) of mutant to wild type template. The FA is calculated as the percentage ratio between the number of mutant DNA molecules (a) and the number of mutant (a) plus wild type (b) molecules (fractional abundance: (a/a+b)).

We used ddPCR supermix for probes (Bio-Rad product number 186-3010), an automated droplet generator (Bio-Rad QX200), automated droplet reader (Bio-Rad QX200), and PCR cycling conditions according to manufacturer instructions, as previously published.⁴⁰ (link) To measure gene-expression level, we used a previously published primer pair and Fam-labeled probe for human ACTA1 mRNA⁶ (link) and used Primer3 software^{51 ,52} to design a primer pair and Fam-labeled probe targeting ACTA1 pre-mRNA at the intron 1-exon 2 splice site. The primer pair and Fam-labeled probe that we used to measure mouse Dmpk was published previously.⁸ (link) The sequences are shown in Table S1. For normalization controls, we used commercially available standard assays for mouse Acta1 (Hex-labeled probe; Bio-Rad unique assay ID dMmuCPE5088325) and mouse general transcription factor 2b (Gtf2b; FAM-MGB; Applied Biosciences assay ID Mm00663250_m1).⁴⁰ (link) After PCR was complete, the plate was loaded into the droplet reader, processed, and analyzed using QuantaSoft software, and total events were quantitated using the mean copy number per μL of duplicate 20-μL assays from individual samples.⁴⁰ (link)

We used ddPCR Supermix for probes (Bio-Rad), an automated droplet generator (Bio-Rad QX200), automated droplet reader (Bio-Rad QX200), and PCR cycling conditions according to the manufacturer's instructions as follows: enzyme activation 95 °C for 10 min (1 cycle), denaturation 94 °C for 30 s followed by annealing/extension at 60 °C for 1 min (40 cycles), enzyme deactivation 98 °C for 10 min (1 cycle), and hold at 4 °C. After PCR was complete, the plate was loaded into the droplet reader, processed/analyzed using QuantaSoft software, and total events quantitated using the mean copy number per microliter of duplicate 20 µl assays from individual samples.
To measure gene expression level, we used previously published primer probe sets for human ACTA1, human DMPK, and mouse Dmpk^{6 (link)}, and standard assays for human GTF2B or mouse Gtf2b (Applied Biosciences, FAM-MGB; assay IDs Hs00976256_m1 and Mm00663250_m1) as normalization controls. The sequence of each PP set that we used to evaluate alternative splicing of INSR, MBNL2, MBNL1, CLASP1, and MAP3K4 is shown in Supplementary Table 10, and the PP sets to identify DMD exons 45–50 and exon 52 deletions with and without exon 51 skipping were published previously^{35 (link)}.

The Bio-Rad SARS-CoV-2 ddPCR Kit (FDA-cleared for COVID diagnosis from nasal swab RNA) was used to measure nucleocapsid transcripts (N1 and N2) of the SARS-CoV2 in Tempus whole blood RNA. cDNA synthesis and ddPCR was accomplished with a one-step RT-ddPCR mix (Bio Rad) added directly to the RNA. A mix of primers and FAM/HEX probes for N1, N2 and human RNase P (RP) was added (Bio Rad). After droplet generation, the thermal cycler reverse transcribed the RNA and then amplified the cDNA. Droplets were read using the Bio Rad QX200 and clusters were gated using the Bio Rad Quantasoft Analysis Pro software. RP was included as a control to confirm proper amplification and reading. Samples generating two or more N1+ or N2+ droplets were considered positive as specified by the manufacturer.