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Online Supplementary Information Transfection and Stable Line Online Supplementary Information Transfection and stable line generation using PC12 cell line Briefly, TrueORFGold expression validated ORF clone of gene of interest with a C-terminal myc-DDK-tag was obtained in a pCMV6 vector (RC205179, OriGene Technologies, Rockville, MD). Mutant constructs were generated by site directed mutagenesis (see Supplementary Table S5 for primer details) of WT ORF as per Stratagene Quikchange II XL kit protocol and used for transformation of XL10Gold E.coli cells. Mutation(s) thus generated were confirmed by PCR-Sanger sequencing. Stable PC12 cells with each of the constructs were generated by Lipofectamine LTX and Plus reagent (Thermo Fisher Scientific Inc.) mediated transfection and G418 selection. Antibodies used for Western Blots Primary antibodies used were Mouse monoclonal anti DDK tag, TA50011-100, OriGene Technologies, Rockville, MD, diluted to 1:2000; Rabbit Polyclonal anti beta actin, ab8227, Abcam, diluted to 1:2000; and Goat polyclonal anti rat CHRNA7, EB08853, Everest Biotech Ltd., UK, diluted to 1:5000. Secondary antibodies used were Goat anti mouse IgG-HRP conjugate, 32430, Thermo Fisher Scientific Inc., diluted to 1:5000; Goat anti Rabbit IgG- HRP conjugate, ab97200, Abcam, diluted to 1:10,000; Donkey anti Goat IgG-HRP conjugate, PA1-28664, Thermo Fisher Scientific Inc., diluted to 1:5000. Supplementary Table S1. List of microsatellite markers used per locus Locus Microsatellite marker D4S1534 SNCA D4S2460 D4S414 D4S405 UCHL1 D4S2974 D4S1592 D12S345 LRRK2 D12S1668 D12S85 Supplementary Table S2. Exome sequencing statistics Subject ID III10 III16 Total reads 95404,772 111819288 Total yield (bp) 9635881972 11293748088 Read length (bp) 101 101 Target regions (bp) 64190747 64190747 Average throughput depth of target regions 150.1X 175.9X Mappable reads (=reads mapped to human genome) 57745458 71600622 Mappable yield (bp) 5641158683 7078061486 % Mappable reads (out of total reads) 60.50% 64.00% On-target reads (=reads mapped to target regions) 24471630 31327254 On-target yield (bp) 1910104406 2537945157 % On-target reads (out of mappable reads) 42.40% 43.80% % On-target reads (out of total reads) 25.70% 28.00% % Coverage of target regions (more than 1X) 96.30% 96.10% Number of on-target genotypes (more than 1X) 6,18,12,286 6,17,11,021 % Coverage of target regions (more than 10X) 86.20% 87.00% Number of on-target genotypes (more than 10X) 55326717 55818677 Median read depth of target regions 27.0X 33.0X Mean read depth of target regions 29.8X 39.5X Supplementary Table S3. List of 320 prioritised variants screened by HaloPlex-targeted sequencing Reference/ Gene Region/ Effect on amino acid Chrom Alternative Change osome Position Allele 1 45813371 G/C TESK2 splicing none 1 100527571 A/G HIAT1 3UTR none 1 114939079 A/G TRIM33 3UTR none DENND2C:NM_198459(1 8Exons):intronic4;DENN D2C:NM_001256404(21E xons):exon7:c.G1189A:p. 1 115164554 C/T DENND2C missense G397S&missense 1 167886728 C/T MPC2 3UTR none 1 201353915 C/G LAD1 splicing none 1 233112095 A/G NTPCR splicing none 1 249105668 G/T SH3BP5L 3UTR none 1 249200497 C/A PGBD2 5UTR none 2 3504418 G/A ADI1 splicing none EML4:NM_019063(23Ex ons):exon23:c.C2845A:p. L949I&missense;EML4:N M_001145076(22Exons):e xon22:c.C2671A:p.L891I 2 42557246 C/A EML4 missense &missense 2 69474669 T/C ANTXR1 3UTR none 2 69702115 G/C AAK1 3UTR none 2 130831551 G/A POTEF 3UTR none 2 132201305 C/T LOC401010 ncRNA none CCTCTCTCTCT CTCTC/CCTCT CCNT2- 2 135625118 CTCTCTC---- AS1 ncRNA none 2 191073581 T/C HIBCH splicing none 2 197065376 A/C HECW2 3UTR none 2 197674132 G/A C2orf66 5UTR none 2 222283642 C/A EPHA4 3UTR none 2 242932049 C/T LINC01237 ncRNA none 2 242932050 C/G LINC01237 ncRNA none 3 73065195 G/A PPP4R2 3UTR none 3 73065197 T/C PPP4R2 3UTR none NCK1:NM_001190796(3 Exons):exon2:c.C138T:p.P 46P&synonymous;NCK1: NM_006153(4Exons):exo n3:c.C330T:p.P110P&syn onymous;NCK1:NM_001 291999(4Exons):exon3:c. C330T:p.P110P&synonym 3 136664528 C/T NCK1 missense ous 3 138048189 G/C NME9 5UTR none CLSTN2:NM_022131(17 Exons):exon3:c.C313T:p. 3 140122551 C/T CLSTN2 missense R105W&missense 3 141879932 TG/T- GK5 3UTR none AAAGGAAGTA 3 142215365 A/AAA-------- ATR splicing none ATR:NM_001184(47Exon s):exon14:c.G2875A:p.V9 3 142269075 C/T ATR missense 59M&missense ANKUB1:NM_00114496 0(6Exons):exon5:c.G1001 3 149485448 C/T ANKUB1 missense A:p.C334Y&missense MED12L:NM_053002(43 Exons):downstream+458;I GSF10:NM_178822(6Exo ns):exon6:c.A7426T:p.R2 476W&missense;IGSF10: NM_001178145(2Exons): exon2:c.A1507T:p.R503W &missense;IGSF10:NM_0 01178146(2Exons):exon2: c.A1363T:p.R455W&miss 3 151154923 T/A IGSF10 missense ense 3 160146591 C/T SMC4 missense SMC4:NM_005496(23Ex ons):exon17:c.C2656T:p.R 886C&missense;SMC4:N M_001002800(24Exons):e xon18:c.C2656T:p.R886C &missense;SMC4:NM_00 1288753(24Exons):exon18 :c.C2581T:p.R861C&miss ense 3 161063031 A/G SPTSSB 3UTR none 3 172349765 G/C NCEH1 3UTR none MCF2L2:NM_015078(30 nonframes Exons):exon1:c.14del- 3 183145747 CTTTT/CT--- MCF2L2 hift AAA:p.K6del 3 186288571 C/T DNAJB11 5UTR none 3 186507309 T/C EIF4A2 3UTR none 3 187440223 C/T BCL6 3UTR none LPP:NM_001167671(11E xons):exon9:c.G1474A:p. G492R&missense;LPP:N M_005578(11Exons):exon 9:c.G1474A:p.G492R&mi ssense;LPP:NM_0011676 72(10Exons):exon8:c.G10 3 188584051 G/A LPP missense 33A:p.G345R&missense 3 195498542 C/T MUC4 missense MUC4:NM_138297(23Ex ons):exon3:c.G362A:p.R1 21Q&missense;MUC4:N M_018406(25Exons):exon 5:c.G13223A:p.R4408Q& missense;MUC4:NM_004 532(24Exons):exon4:c.G5 15A:p.R172Q&missense MUC4:NM_138297(23Ex ons):intronic1;MUC4:NM _004532(24Exons):introni c1;MUC4:NM_018406(25 Exons):exon2:c.T9742C:p. 3 195508709 A/G MUC4 missense S3248P&missense MUC4:NM_138297(23Ex ons):intronic1;MUC4:NM _004532(24Exons):introni c1;MUC4:NM_018406(25 Exons):exon2:c.T4870G:p. 3 195513581 A/C MUC4 missense S1624A&missense CNGA1:NM_000087(11E xons):exon11:c.G1031A:p. R344H&missense;CNGA1 :NM_001142564(10Exons ):exon10:c.G1238A:p.R41 4 47939480 C/T CNGA1 missense 3H&missense FRYL:NM_015030(64Exo ns):exon23:c.A2368G:p.I7 4 48581150 T/C FRYL missense 90V&missense SLC39A8:NM_00113514 6(9Exons):exon7:c.G855T :p.E285D&missense;SLC3 9A8:NM_022154(8Exons) :exon6:c.G855T:p.E285D &missense;SLC39A8:NM _001135148(8Exons):exon 6:c.G654T:p.E218D&miss ense;SLC39A8:NM_0011 35147(11Exons):exon7:c. G855T:p.E285D&missens 4 103189222 C/A SLC39A8 missense e 4 190862104 C/G FRG1 5UTR none 5 11497 A/C unknown none none NLN:NM_020726(13Exon s):exon8:c.G1162A:p.V38 5 65084148 G/A NLN missense 8M&missense DMXL1:NM_001290322( 43Exons):exon18:c.A2596 T:p.S866C&missense;DM XL1:NM_001290321(44E 5 118484637 A/T DMXL1 missense xons):exon18:c.A3115T:p. S1039C&missense;DMXL 1:NM_005509(43Exons):e xon18:c.A3115T:p.S1039 C&missense 5 121297710 CATAT/CAT-- SRFBP1 5UTR none 5 121402284 T/C LOX 3UTR none 5 127614324 T/C FBN2 splicing none 5 132085115 G/C CCNI2 splicing none 5 133915891 C/T JADE2 3UTR none 5 140233291 G/A PCDHA9 3UTR none 5 140574962 A/G PCDHB10 3UTR none PCDHB13:NM_018933(1 CGCTGCTGCC Exons):exon1:c.G1469del- GCCCCAGG/CG CTGCTGCCGCCCCAGG 5 140595161 ---------------- PCDHB13 frameshift :p.L490fs 5 180028968 C/A FLT4 3UTR none 6 84140782 G/C ME1 5UTR none CTATAT/+++++ 6 99720018 +AT FAXC 3UTR none SGK1:NM_001143676(14 Exons):exon14:c.C1565T: p.T522M&missense;SGK 1:NM_001143677(12Exon s):exon12:c.C1364T:p.T45 6 134491422 G/A SGK1 missense 5M&missense;SGK1:NM _001143678(12Exons):exo n12:c.C1322T:p.T441M& missense;SGK1:NM_0012 91995(11Exons):exon11:c. C1148T:p.T383M&missen se;SGK1:NM_005627(12 Exons):exon12:c.C1280T: p.T427M&missense 6 143929340 G/T PHACTR2 5UTR none AKAP12:NM_144497(3E xons):exon2:c.C3701T:p.P 1234L&missense;AKAP1 2:NM_005100(5Exons):ex on4:c.C3995T:p.P1332L& 6 151673521 C/T AKAP12 missense missense 7 856939 C/A SUN1 5UTR none C7orf50:NM_032350(5Ex ons):intronic2;C7orf50:N M_001134395(5Exons):int ronic2;C7orf50:NM_0011 34396(5Exons):intronic2; GPR146:NM_138445(1Ex ons):exon1:c.G829A:p.V2 7 1097980 G/A GPR146 missense 77M&missense 7 5254061 G/A WIPI2 5UTR none 7 5267849 C/G WIPI2 splicing none 7 5965229 C/T RSPH10B 3UTR none ZAN:NR_111918(47Exon s):ncRNA;ZAN:NR_1119 17(48Exons):ncRNA;ZAN :NR_111919(47Exons):nc RNA;ZAN:NM_003386(4 8Exons):exon14:c.T2191C :p.S731P&missense;ZAN: NM_173059(46Exons):ex on14:c.T2191C:p.S731P& 7 100349919 T/C ZAN missense missense ADCK2:NM_052853(8Ex ons):exon6:c.G1662A:p.R 7 140389501 G/A ADCK2 missense 554R&synonymous 7 156432793 A/T C7orf13 ncRNA none 8 11279886 T/G FAM167A 3UTR none ABRA:NM_139166(2Exo ns):exon1:c.C345A:p.S115 8 107782074 G/T ABRA missense R&missense CSMD3:NM_198123(71E xons):exon35:c.T5769A:p. N1923K&missense;CSM D3:NM_052900(69Exons) 8 113418793 A/T CSMD3 missense :exon34:c.T5457A:p.N181 9K&missense;CSMD3:N M_198124(72Exons):exon 36:c.T5649A:p.N1883K& missense COL22A1:NM_152888(6 5Exons):exon3:c.C374G:p 8 139890277 G/C COL22A1 missense .T125R&missense TRAPPC9:NM_031466(2 3Exons):exon22:c.G3419 A:p.R1140H&missense;T RAPPC9:NM_001160372( 23Exons):exon22:c.G3125 8 140744376 C/T TRAPPC9 missense A:p.R1042H&missense 9 139958131 C/A SAPCD2 3UTR none TUBB8:NM_177987(4Ex ons):exon4:c.T932G:p.L31 10 93400 A/C TUBB8 missense 1R&missense 10 855067 C/T LARP4B 3UTR none 10 5904070 G/A ANKRD16 3UTR none IL15RA:NR_046362(5Ex ons):ncRNA;IL15RA:NM _001243539(7Exons):exon 2:c.G129A:p.K43K&syno nymous;IL15RA:NM_001 10 6008154 C/T IL15RA missense 256765(8Exons):exon3:c.
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