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Supplementary Material 1 SUPPLEMENTARY MATERIAL Supplemental Methods CNV validation Validation by quantitative real-time polymerase chain reaction (qPCR) was performed using TaqMan assays (ThermoFisher Scientific, Waltham, MA, USA). Genomic DNA was extracted from one 3-mm dried blood spot,1 diluted 1:10 in water, and amplified using TaqMan Environmental Master Mix in 5 µl reaction volumes. A fragment of the RNaseP H1 RNA gene was co-amplified and used as an internal control (TaqMan Copy Number Reference Assay). Assays were run in quadruplicate on either an Applied Biosystems (ABI) 7900HT or an ABI QuantStudio PCR system (ThermoFisher Scientific). CopyCaller software version 2.0 (ThermoFisher Scientific) was used to analyze the real-time data using relative quantitation (2- ΔΔCt method). The manual Ct threshold was set to 0.2 with the automatic baseline on. CopyCaller software parameters were as follows: the median ΔCt for each experiment was used as the calibrator, wells with an RNaseP Ct > 38 were excluded and the zero copy ΔCt threshold was set to six. The average copy number and a software-generated confidence value were calculated for each subject. Copy-number results with confidence values ≥ 0.95 were considered valid; results with confidence values < 0.95 were re-run in quadruplicate. Targeted sequencing of SHFM candidate genes A custom panel targeting the coding regions and exon-intron boundaries of 49 SHFM candidate genes was designed using the Ion AmpliSeq Designer tool version 2.2.1 with the “standard DNA (225-bp amplicon target sizes)” and “Gene + UTR” options. Two primer pools were used to 2 amplify 1068 amplicons, covering 49 target genes, totaling 174.12 kb. DNA was quantified using an RNaseP TaqMan assay on an ABI 7900HT PCR System (TaqMan RNaseP Control). Libraries were constructed using 500 pg DNA for dried blood spot specimens or 1 ng for buccal specimens, one AmpliSeq primer pool (per reaction), and AmpliSeq library kit 2.0. Amplification was carried out on a GeneAmp PCR System 9700 (ThermoFisher Scientific) for 21 cycles. Reaction-specific primers were removed using FuPa reagent. AmpliSeq PCR products from each subject were ligated to P1 adapters and barcodes using IonXpress Barcode Adapter kits. Barcoded libraries were quantified by qPCR using the Ion Library Quantitation Kit. Purified libraries were sequenced by the Applied Genomics Technologies Core at the Wadsworth Center, New York State (NYS) Department of Health. Purified libraries were diluted to 100 pM and pooled. Template preparation was done on the Ion OneTouch system using the Ion OT 200 Template kit version 2, DL. Amplified Libraries were sequenced on Ion 318 or 318C chips using an Ion Personal Genome Machine sequencer (ThermoFisher Scientific). Specimens were run in three batches. The total aligned output for each of the three runs was 451 megabases over 3.9 million reads (14 test specimens, 4 in duplicate, 318C chip), 1 gigabase over 6.9 million reads (13 test specimens, 318 chip), and 737 megabases over 5 million reads (14 test specimens, 1 in duplicate, 318C chip), respectively. Over all runs, coverage uniformity (defined as base coverage at > 20% mean coverage) was 83.7% and the proportion of on-target bases (proportion of bases mapping to target regions out of total mapped bases per run) was 70.4%, which is consistent with manufacturer specifications. Two NYS specimens from the first run failed (mean read depth 6.5 and 1.2) and were excluded from sequence data analysis and the statistics that follow. The mean (± standard deviation; range) value for number of mapped reads per specimen was 313,681 (±111 023; 122 454 – 808 721), for read depth was 191 X (± 94 3 X; 50 X-588 X), for percentage of bases that had ≥ 20 X coverage was 85.3% (± 12.5%; 41.9%- 92.6%), and for percentage of bases that had ≥ 100 X coverage was 64.2% (± 24.7%; 11.1%- 87.3%). Panel information was imported into Torrent Suite software (version 4.2) for data analysis. Signal processing and base calling were carried out using the default base caller parameters. Sequence data were aligned and mapped to a reference human genome sequence file using the Torrent Mapping Alignment Program (version 2.18) which is optimized for Ion Torrent data. Variants were called using Ion Torrent Variant Caller (version 4.2-18) with selection of default parameters for “PGM - Germ Line - Low Stringency”. Also, the following parameter changes were applied: minimum coverage on either strand = 2 for SNP and INDEL; downsample_to_coverage = 400; do_snp_realignment = 0; mnp_min_cov_each_strand = 2; output_mnv = 1; allow_complex = 1. The variant call format (VCF) files generated for each DNA specimen were merged using the bcftools merge function (bcftools_merge version1.2+htslib-1.2.1; https://github.com/samtools/bcftools). Prior to annotating variants with ANNOVAR,2 variants were decomposed and left-aligned as recommended by ANNOVAR documentation. Multi-allelic variants were decomposed using the vcfbreakmulti function in the vcflib program (https://github.com/vcflib/vcflib), and variants were left-aligned using the normalize function in vt software (version vt-0.57; https://github.com/atks/vt). Variants were then annotated using the table_annovar function in ANNOVAR. Validation of selected sequence variants Sanger sequencing was used to validate the selected sequence variants, as previously described, with minor modifications.3 Primer sequences were selected using Primer Designer Tool 4 (Thermo Fisher Scientific). Primer identification numbers or sequences, and PCR conditions used, are provided in Supplementary Table S2. PCR reactions contained extracted DNA, DNA Master HybProbe master mix (Roche Applied Science, Indianapolis, IN, USA), 1 unit Taq antibody (Clontech, Mountain View, CA, USA), 2.5 mM MgCl2, and 0.2 µM each primer, in a total volume of 25 µl. Standard cycling conditions included an initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 30 seconds, annealing at the specified annealing temperature for 30 seconds, elongation at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. PCR products were cleaned up using ExoSAP-IT (USB Corporation, Cleveland, OH, USA), and sequenced using BigDye Terminator v.3.1 Cycle Sequencing chemistry kits (Thermo Fisher Scientific) on an ABI 3730 DNA Analyzer (Thermo Fisher Scientific). Sequence chromatograms were analyzed using SeqScape version 2.1.1 (Thermo Fisher Scientific) and FinchTV version 1.4.0 (Geospiza, Seattle, WA, USA). 5 Supplementary Figure S1 Infinium HumanOmni2.5-4 microarray results for the chromosome 10q24 region. Images of copy-gain region for patient 1, and image of the same 10q24 region for a control subject. Bottom panel: genes located in or near the copy-gain region. 6 Supplementary Figure S2 Infinium HumanOmni2.5-4 microarray results for the chromosome 17p13.3 region. Images of copy-gain regions for patients 2, 3, and 4, and image of the same 17p13.3 region for a control subject. Bottom panel: genes located in or near the copy-gain region. 7 Supplementary Figure S3 Infinium HumanOmni2.5-4 microarray results for the chromosome 17q25 region. Images of copy-loss region for patient 16, and image of the same 17q25 region for a control subject. Bottom panel: genes located in or near the copy-loss region. 8 TP63 p.R225H TP63 p.R225L TP63 p.P417T EVX2 p.A472T HOXD12 p.N237T HOXD11 p.G245D HOXD10 p.L57P HOXD3 p.G42S HOXD1 p.G218R FGFR1 p.P283S 9 ROR2 p.D895G POLL p.E498K CDH3 p.R175W CDH3 p.M269L Supplementary Figure S4 Electropherograms from Sanger sequencing showing 14 validated, non-synonymous mutations in candidate genes for split hand/foot malformation. The mutations were detected in 11 cases with split hand/foot malformation. 10 Supplementary Figure S5 Evolutionary conservation of the tumor protein p63 (TP63) amino acid sequence. (a) The conserved R225 amino acid in the DNA-binding domain. (b) Proline-rich region showing conservation of the proline at position 417. Prolines at other positions are marked in blue. 11 Supplementary Figure S6 Expression of genes located in the chromosome 17q25 region (chr17:73105000-73428037; GRCh37/hg19 assembly) in human limb buds at embryonic day 44. Gene expression (RNA-seq) data were generated by Cotney et al.4 To view the gene expression data in the University of California at Santa Cruz (UCSC) Genome Browser,5 RNA-seq BAM files were converted to bigWig files using the Galaxy software platform.6-8 12 Supplementary Figure S7 Expression of genes located in the LBX1-FGF8 region (chr10:102900967-103550000; GRCh37/hg19 assembly) in human limb buds at embryonic day 44. Gene expression (RNA-seq) data were generated by Cotney et al.4 13 Supplementary Figure S8 Expression of genes located in the ABR-TUSC5 region (chr17:900000-1250000; GRCh37/hg19 assembly) in human limb buds at embryonic day 44. Gene expression (RNA-seq) data were generated by Cotney et al.4 14 Supplementary Figure S9 Alignment of predicted enhancers with peaks of histone H3K27ac modifications in the chromosome 10q24 region in human limb buds. The depicted region spans chr10:102900967-103550000 (GRCh37/hg19 assembly). Shaded areas 15 show the alignment of the predicted enhancers, previously determined to be conserved non-coding elements,9,10 with peaks of evolutionary conservation (based on multiple alignment of the genomes of 100 vertebrates using the PhyloP method) and peaks of histone H3K27ac modification detected by chromatin immunoprecipitation in human limb buds at embryonic day 33 (E33), E41, E44, and E47. Genomic coordinates for the predicted enhancers are provided in Supplementary Table S9. The histone H3K27ac modification (ChIP-seq) data were generated by Cotney et al.4 16 Supplementary Figure S10 Evolutionarily conserved gene order at the 17p13.3 region associated with split hand/foot malformation.
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