Sequencing analysis software 6

Primers were designed for two amplicons to cover the maternal (NM_000112.3(SLC26A2):c.-26 + 2T>C) and paternal (NM_000112.3(SLC26A2):c.1957T>A) variants (Supplementary Table S2). The PCR amplifications were run in a 25-µL reaction with the same PCR mix as for RP-PCR and with a final concentration of the forward and reverse primer of 1.0 µM of each. The template was 20 ng single-cell WGA-DNA, 20 ng WGA-DNA pool from all EVTs, or 0.5 ng gDNA. The PCR program was 10 min at 95°C, 33 cycles of 95°C for 30 s, 58°C (for SLC26A2 c.-26 + 2T>C) or 59°C (for SLC26A2 c.1957T>A) for 30 s and 72°C for 30 s, followed by 7 min final extension at 72°C and a hold at 4°C. Sanger sequencing was performed with SLC26A2 c.-26 + 2T>C forward primer, and SLC26A2 c.1957T>A reverse primer, at MOMA, Aarhus University Hospital, Denmark. Data analysis was done using Sequencing Analysis Software 6 (Applied Biosystems, Thermo Fisher Scientific, US).

A primer set was designed to cover the family specific variant NM_004006.2(DMD):c.4358_4359insAATA. The forward- and reverse primers were tagged with a universal M13-primers sequence for Sanger Sequencing in both directions (Supplementary Table S3). A FAM labeled forward primer was used for fragment length analysis using ABI 3500. The PCR was run in a 25-µL reaction volume with 1 X Q-solution, 1 X PCR buffer, 1 U of HotStarTaq DNA polymerase (Qiagen, DE), 0.2 mM dNTP and a final concentration of the forward and reverse primers of 0.5 uM each. The template was 1 µL of WGA-DNA from individual EVTs or a pool of WGA-DNA. Thermal cycling was performed with 15 min at 95°C followed by 35 cycles of 94°C for 1 min, 60°C for 30 s and 72°C for 1 min, and terminated by a final extension at 72°C for 10 min. Sanger Sequencing was conducted at the Department of Molecular Medicine, Aarhus University Hospital. Sanger sequencing data was accessed using Sequencing Analysis Software 6 (Applied Biosystems, Thermo Fisher Scientific, US).

For the detection of FGFR3-related disorders, amplicons were designed to cover the FGFR3 variant of case 1a (NM_000142.4(FGFR3):c.1620C>G and case 1b (NM_000142.4(FGFR3):c.1138G>A). One µL single-cell WGA-DNA product from cells of fetal origin was used as input for PCR amplification using 5 U AmpliTaq Gold™ 360 DNA Polymerase with 10X AmpliTaq Gold^® 360 Buffer, 4 μL GC Enhancer (Thermo Fisher Scientific, US), 1.5 mM MgCl₂ (25 mM) and 2 mM dNTP (10 mM solution of dNTP containing 2.5 mM each of dATP, dCTP, dGTP and dTTP) in a final reaction volume of 25 µL. The forward- and reverse primers were added for a final concentration of 0.2–2 µM (Supplementary Table S1). Thermal cycling was performed with an initial denaturation at 95°C for 10 min followed by 33 cycles of 95°C for 30 s, primer specific annealing temperature for 30 s and 72°C for 45 s, and with a final extension at 72°C for 7 min before a hold at 4°C using a Veriti™ 96-Well Thermal Cycler (Thermo Fisher Scientific, US). The PCR products were analyzed by capillary electrophoresis using an ABI 3500 (Thermo Fisher Scientific, US) for size and purity. The reverse primers (2 pmol/μL) were used for Sanger Sequencing at the Department of Molecular Medicine (MOMA), Aarhus University Hospital, Denmark. Data analysis was performed using Sequencing Analysis Software 6 (Applied Biosystems, Thermo Fisher Scientific, US).