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Development and evaluation of a droplet digital PCR assay for 8p23 beta- defensin cluster copy number determination Tingting Wen

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Development and evaluation of a droplet digital PCR assay for 8p23 beta- defensin cluster copy number determination

Tingting Wen

1

, Xianghong Zhang

2*

, Christoph Lippuner

3

, Marcel Schiff

3

, and Frank Stuber

3

1

Department of Anesthesiology, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China

2

Department of Cardiac Intensive Care Unit, the Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Heath, Hangzhou, Zhejiang, China

3

Department of Anesthesiology and Pain Medicine, Bern University Hospital, Inselspital, Bern, Switzerland

*Corresponding author

Email: xianghong_zhang@zju.edu.cn

(2)

Supplementary Fig. 1 Bar plots represent events number for ddPCR reactions using

(a) 80 ng and (b) 2.5 ng DNA template. Absolute events number was labeled above

the bar. Red arrows indicate too few (a) negative and (b) positive events which result

in ambiguous or inconsistent CN determination. Light blue: DEFB4 positive; Dark

blue: DEFB4 negative; Light green: RPP30 positive; Dark green: RPP30 negative

(3)

Supplementary Table 1 DEFB CN determined by ddPCR in 283 healthy Europeans

Sample ID Raw CN-1 Raw CN-2 CN-1 CN-2 ddPCR-CN MLPA-CN

RC001 4.85 5.12 5 5 5 5

RC002 6.08 6.09 6 6 6 6

RC003 3.87 3.84 4 4 4 4

RC004 4.04 4.02 4 4 4 4

RC005 5.25 4.81 5 5 5 5

RC006 6.04 6.12 6 6 6 6

RC007 4.94 5.15 5 5 5 5

RC008 5.96 5.94 6 6 6 6

RC009 2.95 3.05 3 3 3 3

RC010 4.8 4.99 5 5 5 5

RC011 6.88 7.12 7 7 7 7

RC012 4.85 4.89 5 5 5 5

RC013 3.86 4.04 4 4 4 4

RC014 2.94 2.95 3 3 3 3

RC015 7.04 6.9 7 7 7 7

RC016 4.93 4.97 5 5 5 5

RC017 2.93 3 3 3 3 3

RC018 5 4.81 5 5 5 5

RC019 3.97 3.94 4 4 4 4

RC020 5.92 6.16 6 6 6 6

RC021 5.03 4.98 5 5 5 5

RC022 4.16 3.97 4 4 4 4

RC023 6.94 7.09 7 7 7 7

RC024 6.03 6.02 6 6 6 6

RC025 5.08 5.17 5 5 5 5

RC026 3.01 3.01 3 3 3 3

RC027 3.88 4.05 4 4 4 4

RC028 4.91 5.04 5 5 5 5

RC029 4.96 5.14 5 5 5 5

RC030 3 2.88 3 3 3 3

RC032 4 3.9 4 4 4 4

RC033 4.78 4.8 5 5 5 5

RC034 4.06 3.99 4 4 4 4

RC035 5.21 4.91 5 5 5 5

RC036 4.05 4.1 4 4 4 4

RC037 5.15 4.93 5 5 5 5

RC038 3.97 4.14 4 4 4 4

RC039 5.02 5.01 5 5 5 5

RC040 4.05 3.95 4 4 4 4

RC041 4.03 3.97 4 4 4 4

RC042 3.09 3.04 3 3 3 3

RC043 3.95 4.01 4 4 4 4

RC044 4.95 5.02 5 5 5 5

RC045 5.08 5.13 5 5 5 5

RC046 4 4.19 4 4 4 4

(4)

RC047 2.99 3.22 3 3 3 3

RC048 6.26 5.94 6 6 6 6

RC050 5.77 6.15 6 6 6 6

RC051 3.98 4.05 4 4 4 4

RC052 3.98 3.99 4 4 4 4

RC053 3.98 4.09 4 4 4 4

RC054 3.96 3.92 4 4 4 4

RC056 3.02 2.96 3 3 3 3

RC057 4.01 3.97 4 4 4 4

RC058 5.81 5.91 6 6 6 6

RC059 5.1 4.93 5 5 5 5

RC060 3.94 4.08 4 4 4 4

RC061 5.07 4.78 5 5 5 5

RC062 5.09 5 5 5 5 5

RC064 5.03 4.8 5 5 5 5

RC065 6.93 7.2 7 7 7 7

RC066 3.03 3.04 3 3 3 3

RC067 3.03 3.13 3 3 3 3

RC068 4.06 4.2 4 4 4 4

RC069 4.96 5.1 5 5 5 5

RC070 3.81 4.03 4 4 4 4

RC071 8.01 8.04 8 8 8 8

RC072 5.26 4.97 5 5 5 5

RC073 2.87 2.88 3 3 3 3

RC074 4.95 5.13 5 5 5 5

RC075 4.05 3.97 4 4 4 4

RC076 3.94 3.95 4 4 4 4

RC077 3.94 3.98 4 4 4 4

RC078 4.08 4.08 4 4 4 4

RC079 5.01 5 5 5 5 5

RC080 2.95 3 3 3 3 3

RC081 5.06 5.01 5 5 5 5

RC082 4.04 3.92 4 4 4 4

RC083 5.07 5.03 5 5 5 5

RC084 3.97 3.97 4 4 4 4

RC085 3.94 4.07 4 4 4 4

RC087 3.02 2.96 3 3 3 3

RC089 2.97 2.98 3 3 3 3

RC090 5.75 5.92 6 6 6 6

RC092 5.92 5.85 6 6 6 6

RC094 4.96 4.99 5 5 5 5

RC095 3.89 3.83 4 4 4 4

RC096 4.79 4.8 5 5 5 5

RC097 4.91 4.99 5 5 5 5

RC098 3.92 3.9 4 4 4 4

RC099 6.1 5.94 6 6 6 6

RC100 5.21 4.88 5 5 5 5

RC102 5.05 5.05 5 5 5 5

(5)

RC103 3.05 3.05 3 3 3 3

RC104 6.09 6.25 6 6 6 6

RC105 4.13 4.09 4 4 4 4

RC106 4.03 4.1 4 4 4 4

RC107 2.92 3.08 3 3 3 3

RC108 3.96 4.01 4 4 4 4

RC109 5.69 6.02 6 6 6 6

RC110 4.89 5.21 5 5 5 5

RC111 4.04 3.85 4 4 4 4

RC112 5.24 5.03 5 5 5 5

RC113 1.9 1.98 2 2 2 2

RC114 4.98 5.09 5 5 5 5

RC115 4.93 4.78 5 5 5 5

RC116 4.08 3.99 4 4 4 4

RC117 2.98 2.92 3 3 3 3

RC118 3.76 3.91 4 4 4 4

RC119 5.08 4.95 5 5 5 5

RC120 2.93 2.94 3 3 3 3

RC121 6.81 7.08 7 7 7 7

RC122 4.14 3.88 4 4 4 4

RC123 3.9 4.04 4 4 4 4

RC124 6.06 6.1 6 6 6 6

RC125 3.85 3.82 4 4 4 4

RC126 4.04 4.03 4 4 4 4

RC127 3.93 4.16 4 4 4 4

RC128 3.07 2.97 3 3 3 3

RC129 3.09 3.1 3 3 3 3

RC130 5 4.92 5 5 5 5

RC131 6.03 5.99 6 6 6 6

RC132 4.08 4.17 4 4 4 4

RC133 4 3.9 4 4 4 4

RC134 4.86 4.91 5 5 5 5

RC135 3.95 3.72 4 4 4 4

RC136 2.03 2.12 2 2 2 2

RC137 4.96 4.84 5 5 5 5

RC138 4.01 3.98 4 4 4 4

RC139 6.16 5.67 6 6 6 6

RC140 3.9 3.89 4 4 4 4

RC141 3.1 2.84 3 3 3 3

RC142 4.05 4.13 4 4 4 4

RC144 3.96 4.11 4 4 4 4

RC145 4.19 3.77 4 4 4 4

RC146 3.92 3.88 4 4 4 4

RC147 2.92 3.08 3 3 3 3

RC148 4.04 4.06 4 4 4 4

RC149 5.76 5.93 6 6 6 6

RC150 5.03 4.95 5 5 5 5

RC153 3 2.9 3 3 3 3

(6)

RC154 5.99 5.91 6 6 6 6

RC155 3.89 3.95 4 4 4 4

RC156 5.12 4.91 5 5 5 5

RC157 4 4.14 4 4 4 4

RC159 3.9 3.91 4 4 4 4

RC160 3.94 3.94 4 4 4 4

RC161 5 5.13 5 5 5 5

RC162 5 4.76 5 5 5 5

RC163 2.97 2.88 3 3 3 3

RC164 4.01 3.96 4 4 4 4

RC165 6.29 5.97 6 6 6 6

RC166 3.01 2.91 3 3 3 3

RC167 6.07 6.09 6 6 6 6

RC168 5.85 5.75 6 6 6 6

RC169 5.08 4.91 5 5 5 5

RC170 3.76 3.96 4 4 4 4

RC171 4.04 3.82 4 4 4 4

RC172 5.94 6.06 6 6 6 6

RC173 4.99 4.98 5 5 5 5

RC174 5 5.03 5 5 5 5

RC175 3.96 3.97 4 4 4 4

RC176 3.03 3.01 3 3 3 3

RC177 2.99 3.03 3 3 3 3

RC178 9.18 8.79 9 9 9 9

RC179 6.21 5.83 6 6 6 6

RC180 4.02 3.92 4 4 4 4

RC181 3.87 4 4 4 4 4

RC182 3.98 3.97 4 4 4 4

RC183 5.13 4.96 5 5 5 5

RC184 4.97 5.06 5 5 5 5

RC185 5.07 5.07 5 5 5 5

RC186 4.9 5 5 5 5 5

RC187 4.97 4.99 5 5 5 5

RC188 4.06 3.93 4 4 4 4

RC189 4.09 4.03 4 4 4 4

RC190 4.97 4.91 5 5 5 5

RC191 4.06 3.77 4 4 4 4

RC192 4.01 4.05 4 4 4 4

RC193 5.24 5.04 5 5 5 5

RC194 5.01 5.04 5 5 5 5

RC195 4.92 4.83 5 5 5 5

RC196 1.94 1.93 2 2 2 2

RC197 5.05 4.97 5 5 5 5

RC198 3.85 4.03 4 4 4 4

RC199 4 4.05 4 4 4 4

RC200 4.05 3.87 4 4 4 4

RC201 4 3.94 4 4 4 4

RC202 6.02 6.12 6 6 6 6

(7)

RC203 4.81 5.04 5 5 5 5

RC204 5.01 4.89 5 5 5 5

RC205 4.17 4 4 4 4 4

RC206 4.08 4 4 4 4 4

RC207 4.98 5.1 5 5 5 5

RC208 3.91 3.96 4 4 4 4

RC209 4.87 4.98 5 5 5 5

RC210 5.24 4.85 5 5 5 5

RC211 5.01 4.96 5 5 5 5

RC212 3.09 2.95 3 3 3 3

RC213 3.05 3.1 3 3 3 3

RC214 4.11 3.82 4 4 4 4

RC215 4.08 3.96 4 4 4 4

RC216 2.96 2.9 3 3 3 3

RC217 4.99 4.84 5 5 5 5

RC218 3.94 3.91 4 4 4 4

RC219 7.17 6.88 7 7 7 7

RC220 4.9 5.16 5 5 5 5

RC221 4.91 4.92 5 5 5 5

RC222 3.94 3.83 4 4 4 4

RC223 4.06 3.9 4 4 4 4

RC224 4.16 4.15 4 4 4 4

RC225 5.03 4.91 5 5 5 5

RC226 4.24 3.82 4 4 4 4

RC227 4.08 4.14 4 4 4 4

RC228 4.15 3.88 4 4 4 4

RC229 2.95 2.95 3 3 3 3

RC230 2.84 3.09 3 3 3 3

RC231 4.05 4 4 4 4 4

RC232 4.05 4 4 4 4 4

RC233 4.05 4.03 4 4 4 4

RC234 3.01 2.95 3 3 3 3

RC235 3.94 4 4 4 4 4

RC236 4.12 3.92 4 4 4 4

RC237 5.22 5.16 5 5 5 5

RC238 4.02 4.1 4 4 4 4

RC239 3.91 3.88 4 4 4 4

RC240 5.96 5.83 6 6 6 6

RC241 2.95 2.93 3 3 3 3

RC242 4.17 3.87 4 4 4 4

RC243 2.99 2.89 3 3 3 3

RC244 4.9 4.94 5 5 5 5

RC245 4 4.17 4 4 4 4

RC246 5.06 5.03 5 5 5 5

RC247 4.03 4.06 4 4 4 4

RC248 3.72 3.87 4 4 4 4

RC249 3.89 3.99 4 4 4 4

RC250 4.81 4.95 5 5 5 5

(8)

RC251 3.03 3.02 3 3 3 3

RC252 4.88 4.93 5 5 5 5

RC253 5.06 4.9 5 5 5 5

RC254 4 4.16 4 4 4 4

RC255 6.07 6.07 6 6 6 6

RC256 1.97 1.99 2 2 2 2

RC257 5.14 5.1 5 5 5 5

RC258 4.95 5.02 5 5 5 5

RC259 3.95 4.11 4 4 4 4

RC260 8.17 8.18 8 8 8 8

RC261 3.02 2.97 3 3 3 3

RC262 6.08 5.94 6 6 6 6

RC263 3.81 4.09 4 4 4 4

RC264 6.93 7.3 7 7 7 7

RC266 5.75 5.86 6 6 6 6

RC267 3.96 3.98 4 4 4 4

RC268 3.99 3.95 4 4 4 4

RC269 3.84 3.83 4 4 4 4

RC270 2.01 1.96 2 2 2 2

RC272 6.19 6.09 6 6 6 6

RC273 4.97 5.05 5 5 5 5

RC274 3.99 4.08 4 4 4 4

RC275 4.95 4.98 5 5 5 5

RC276 4.96 4.89 5 5 5 5

RC277 5.12 4.98 5 5 5 5

RC278 5.14 4.81 5 5 5 5

RC279 3.86 4.01 4 4 4 4

RC280 4.02 3.95 4 4 4 4

RC281 4.07 4 4 4 4 4

RC282 2.99 3.04 3 3 3 3

RC284 3.76 3.94 4 4 4 4

RC285 3.78 3.89 4 4 4 4

RC286 4 3.95 4 4 4 4

RC287 5.02 5.01 5 5 5 5

RC288 4.88 5.11 5 5 5 5

RC289 6.07 5.92 6 6 6 6

RC290 4.03 4.05 4 4 4 4

RC291 3.85 3.86 4 4 4 4

RC292 4.93 4.93 5 5 5 5

RC293 4.02 4.25 4 4 4 4

RC294 5.23 4.87 5 5 5 5

RC295 5.79 5.99 6 6 6 6

RC296 6.17 5.92 6 6 6 6

RC297 5.82 5.92 6 6 6 6

RC298 5.07 5.02 5 5 5 5

RC299 3.01 3.05 3 3 3 3

Raw CN values for each sample were included. Known CNs were previously

determined by MLPA.

(9)

Supplementary Table 2 DEFB CN determined by qPCR in 48 healthy Europeans

Sample ID qPCR-CN-1 qPCR-CN-2 ddPCR-CN

RC113 2 2 2

RC136 2 2 2

RC196 2 2 2

RC256 2 2 2

RC270 2 2 2

RC056 3 3 3

RC101 3 3 3

RC103 3 3 3

RC129 3 3 3

RC163 3 2 3

RC230 3 3 3

RC241 3 3 3

RC243 3 3 3

RC019 4 3 4

RC052 4 4 4

RC070 4 4 4

RC135 4 4 4

RC170 3 3 4

RC198 4 3 4

RC214 4 3 4

RC284 4 3 4

RC016 5 4 5

RC035 4 4 5

RC079 5 4 5

RC102 5 4 5

RC115 5 4 5

RC134 4 4 5

RC185 4 4 5

RC237 5 4 5

RC006 5 5 6

RC048 5 4 6

RC050 5 5 6

RC104 5 4 6

RC240 5 4 6

RC272 6 5 6

RC289 5 5 6

RC297 6 5 6

RC011 6 5 7

RC015 6 6 7

RC023 6 5 7

RC065 6 5 7

RC219 6 5 7

RC264 6 5 7

RC071 7 6 8

RC158 7 6 8

RC260 7 6 8

(10)

RC178 8 7 9

CNs determined by ddPCR were included and regarded as true. Green samples:

consistent CNs between replicates and concodant with ddPCR; yellow samples:

consistent CNs between replicates but underestimated compared to ddPCR; red

samples: inconsistent CNs between replicates.

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