FastDigest Eco47I

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	We first digested the DNA with FastDigest Eco47I restriction enzyme (Thermo Scientific). The final reaction contained 33 ng digested DNA, 900 nM of each primer, and 250 nM of each probe. Twenty microliters of the reaction was loaded into a droplet generator cartridge. Droplets were generated following the manufacturer’s suggested protocol. Cycling conditions were 95° for 10 min, followed by 40 cycles of 94° for 30 sec and 60° for 1 min, and a final 10 min at 98°.

Protocol tips

We first digested the DNA with FastDigest Eco47I restriction enzyme (Thermo Scientific). The final reaction contained 33 ng digested DNA, 900 nM of each primer, and 250 nM of each probe. Twenty microliters of the reaction was loaded into a droplet generator cartridge. Droplets were generated following the manufacturer’s suggested protocol. Cycling conditions were 95° for 10 min, followed by 40 cycles of 94° for 30 sec and 60° for 1 min, and a final 10 min at 98°.

Publication protocol

"We genotyped a group of Swedish and American samples including cases, obligate/potential carriers, and controls by using TaqMan assays designed based on Del-1 and Del-2 sequences from EquCab2.0. As reference assay we used RNAseP (Table S6). Reactions contained 20 ng DNA that was not digested with any restriction enzyme but otherwise analyzed as described below, using the ddPCR platform.

We also genotyped a random set of Shetland ponies (94 individuals) and individuals with known phenotypes by using TaqMan assays designed from BAC-derived consensus contigs on a droplet ddPCR instrument. In this experiment we used an assay targeting myostatin (MSTN) as a reference, three assays targeting Del-1 sequences, and three assays targeting Del-2 sequences (Table S6). The sequences of Del-1 and Del-2 are highly polymorphic, which may affect assay performance. Thus, we consulted Illumina read alignments to place primers and probes in nonpolymorphic regions and also designed three assays for each deletion to use alternative assays in case genotyping results appeared unreliable.

The ddPCR experiments were performed using the Bio-Rad QX100 ddPCR platform. We first digested the DNA with FastDigest Eco47I restriction enzyme (Thermo Scientific). The final reaction contained 33 ng digested DNA, 900 nM of each primer, and 250 nM of each probe. Twenty microliters of the reaction was loaded into a droplet generator cartridge. Droplets were generated following the manufacturer’s suggested protocol. Cycling conditions were 95° for 10 min, followed by 40 cycles of 94° for 30 sec and 60° for 1 min, and a final 10 min at 98°. The PCR plate was transferred to QX100 droplet reader for reading and the data were analyzed using the software QuantaSoft. For ambiguous events, we used the ellipse, rectangle, and Lasso threshold in order to adjust the classification of clusters. To classify genotypes of individuals we considered a range for the measured copy numbers as follows: two copies (1.7–2.3), one copy (0.7–1.3), and for null copy we did not find any outliers to adjust the range.

We performed ANOVAs to test the association between height, deletion genotypes and sex by using the following models:

Height = genotype_class + sex (1)
where genotype_class consists of Del_carriers (Del-1/wild type (WT) and Del-2/WT together) and WT/WT.

Height = genotype + sex (2)
where genotype consists of Del-1/WT, Del-2/WT, and WT/WT."

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Manufacturer protocol

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