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Supplementary data Extraction and analysis of cell free fetal DNA in maternal plasma Following discussion with the mother, 20ml of blood was drawn into standard EDTA tubes. The blood was transferred to the Regional Genetics Laboratory for further processing within 24 hours of blood draw. Materials and Methods Sample Processing Plasma was separated from the blood cells by centrifugation at 1500 g for 10 minutes. The supernatant was then transferred to fresh tubes ensuring that the buffy coat remained intact. The plasma was then centrifuged at 16000 g for 10 minutes to remove any remaining cells, transferred into 2 ml Lo-Bind tubes (Eppendorf) and stored at -80°C until DNA extraction. DNA Extraction DNA was extracted from 400µl or 800µl of plasma using the QIAamp MinElute Virus Spin Kit (Qiagen) according to manufacturer’s instructions, and was eluted into a final volume of 75µl AVE elution buffer. PCR and restriction digest To amplify exon 8 of the FGFR3 gene the following primers were used: For5’GTG TAT GCA GGC ATC CTC ATG TAC 3’; Rev-5’ GGA GAT CTT GTG CAC GGT GG 3’. PCR was carried out on 5, 10 or 20µl of ffDNA using 1X PCR Buffer II, 2mM MgCl2, 200µM of each dNTP, 20pmol of each primer, and 2.5U amplitaq gold in a final volume of 50µl. Touchdown PCR conditions were used as follows: one cycle of 95°C for five minutes, followed by seven cycles of 95°C for 30 s, 62°C for 30 s (decreasing by 1°C per cycle), and 72 °C for 45 s; then 50 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 30 s and finished with 72°C for 10 minutes to complete the extension reaction. Restriction digest of the PCR product was carried out using BsrG1 at 37°C for two hours. PCR to amplify a 132bp region of exon 8 containing the mutation causative for achondroplasia was carried out on 5, 10 or 20µl of DNA extracted from 400µl or 800µl of plasma, as well as on genomic DNA from an unaffected and a positive control. On an unaffected DNA sample, restriction digest of the PCR product with BsrG1 will not cut the DNA, giving rise to a single 132bp fragment, whereas if the mutation is present a BsrG1 restriction site is created, and digestion produces fragments of 132bp, 112bp and 20bp (Figure 3). The majority of the DNA appears unaffected as it is contributed by maternal cell free DNA (cfDNA) and also by the unaffected fetal allele. The fetal DNA constitutes only a small fraction of the cfDNA; increasing amounts of plasma allow for easier visualisation of the band indicating the mutated allele.

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