Prague, 11 September 2019r Magdalena Marcińska, Agnieszka Parys-Proszek, Maria Wróbel, Tomasz Kupiec Internal validation study of the Applied Biosystems TM NGM Detect TM kit
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Prague, 11 September 2019rMagdalena Marcińska, Agnieszka Parys-Proszek, Maria Wróbel, Tomasz Kupiec
Internal validation study of the Applied Biosystems TM NGM DetectTM kit
Workflow
Magnetic extraction
Organic extraction(bones,teeth)
Quantifiler Trio
NGMDetectGlobalFilerNGM
Electrophoresis3500xL
X2
SGM, Identifiler - 140 ’
Identifiler Plus, NGM, NGMSelect - 117’
GlobalFiler – 59’
NGMDetect –34’
Amplification time
Main characteristics
• Higher sample input volume:
NGMDetect: 15µl 500pg
NGMSelect: 10µl 1000 pg
GlobalFiler: 15µl 1000 pg
• Six color approach• 16 autosomal STRs, Amelogenin, additional Yindel• 7 mini-STRs below 230 bp• Internal quality control system
• Newly designed primers except for D12S391, THO1 primers – share with GlobalFiler and NGMSelect kits
• Rearrangement of the loci - Double typing procedure – complementary marker positions
Rearrangement of loci in NGM-Detectand NGM – SElect kits
Optimized positioning of SE33 with lower molecular weight range
www.thermofisher.com
New features: IQC, Yindel
IQC system - two synthetic DNA fragment: LMW (IQCS) and HMW (IQCL)- quality control of PCR and sample’s quality check
HMW (IQCL)LMW (IQCS)
Y INDEL
Internal validation experiments
- Sensitivity,- Reproducibility,- Accuracy,- Mixture study,- Casework samples analysis – bones samples,
Threshold set-up:LOD, LOQ, Stochastic threshold, Stutter ratio,
Procedure validation performed according to SWGDAM guidelines
Sensitivity – 500 pg
-Three replicates of control DNA 007 in serial dilutions
Sensitivity - 250 pg
Sensitivity – 125 pg
Sensitivity - 63 pg
Sensitivity - 31 pg
Sensitivity - 16 pg
Sensitivity - 7 pg
Sensitivity - stochastic effect (31 pg)
Sensitivity - stochastic effect (31 pg)
Sensitivity - stochastic effect (31 pg) Sensitivity - stochastic effect (31 pg)
Sensitivity - summary
PHR > 60%
No results
PHR < 60%
Mixture proportion
Control 007 Control 9947
1 1
1 2
1 5
1 8
1 10
1 20
1 30
1 50
1 1
2 1
5 1
8 1
10 1
20 1
30 1
50 1
NGM Detect Control DNAXY 007
Control DNAX 9947A
D2S1338 20,23 19,23
SE33 17,25.2 19,29.2
D16S539 9,10 11,12
D18S51 12,15 15,19
TH01 7,9.3 8,9.3
D12S391 18,19 18,20
D3S1358 15,16 14,15
FGA 24,26 23,24
Y-indel 2
Amelogenina X,Y X
VWA 14,16 17,18
D21S11 28,31 30
D18S51 12,15 15,19
D2S441 14,15 10,14
D8S1179 12,13 13
D19S433 14,15 14,15
D22S1045 11,16 11,14
D10S1248 12,15 13,15
Mixture analysisMixture analysis
Mixture analysis
1:1
Mixture analysis Mixture analysis
Mixture analysis
Mixture analysis
Mixture analysis
STR n-4 n-2 n+4 n-3 n+3D2S1338 0,1481 - - - -
SE33 0,1928 0,0798 0,085 - -D16S539 0,1461 - - - -D18S51 0,1593 - 0,055 - -TH01 0,0507 - - - -
D12S391 0,1963 - - - -D3S1358 0,1533 - - - -
FGA 0,1375 - 0,0476 - -VWA 0,1744 - - - -
D21S11 0,1408 - 0,0408 - -D1S1656 0,1751 0,0357 0,0509 - -D2S441 0,0889 - - - -D8S1179 0,1276 - 0,0303 - -D19S433 0,1271 - - - -D22S1045 - - - 0,1645 0,0919D10S1248 0,1588 - - - -
NGM DetectInj. 20”
NGM DetectInj. 10”
LOQ600 (B)
500 (G,Y, P)1000 (R)
250 (B, G,Y, P)450 (R)
LOD 250 (B, G,Y, P)500 (R)
120 (B, G,Y, P)200 (R)
Stochastic 2196 1014
Stutter:Thresholds LOD, LOQ:
Efficiency comparison of GlobalFiler/NGM and NGM Detect kits in different types of
casework samples.
Casework sample analysis – bone, teeth samples
12 bone samples and 5 teeth samples with different degradation level
0
20
40
60
80
100
1.00 1.25 1.30 1.50 2.59 3.84 3.97 4.40 5.95 7.99 9.46 10.80 15.61 18.90 22.70 27.00 125.00
% t
yped
mar
kers
Degradation Index
samples with DNA input below 0,5ng
NGM Detect
Casework samples analysis – bone sampleGlobalFiler
NGM-Detect
GlobalFiler
Casework samples analysis – tooth DI=8
D16S539SE33D2S1338
D2S441D10S1248
NGM Detect
Casework samples analysis – tooth DI=8
D2S441D10S1248
D16S539SE33
D2S1338
• NGM Detect = 11 STRs
• GlobalFiler = 13 STRs
• NGM Detect & GlobalFiler = 18 STRs
NN & mother
LR=263
LR=85
LR=12135
Casework samples analysis – tooth DI=8
Statistical parameteres in double amplification
GlobalFiler
Casework samples analysis – petrous bone DI=19
D2S441D10S1248
D16S539SE33
D2S1338
NGM Detect
Casework samples analysis – petrous bone DI=19
D2S441D10S1248
D16S539SE33
D2S1338
• NGM Detect = 11 STRs
• GlobalFiler = 14 STRs
• NGM Detect & GlobalFiler = 18 STRs
Casework samples analysis – petrous bone DI=19
Increased number of positively typed markersin double amplification strategy
Background artifact peaks in analyses of old bones
Background artifact peaks in analyses of old bones
New formulation: NGM – Detectchanges to the IQC large and THO1 primers
NGM-Detect14 STRs
Global Filer12 STRs
NGM-Detect& GF
14 STRs
New formulation: NGM – Detect –changes to the IQC large and THO1 primers
NGM-Detect10 STRs
Global Filer4 STRs
NGM-Detect& GF
12 STRs
1. Short amplification time2. Higher input volume3. Rearrangement of the loci (e.g. D2S1338, SE33)4. Quality control markers (IQCS, IQCL) - a good control
of amplification proccess
• Comparable results of NGM Detect and GlobalFiler,NGM with good quality samples
• NGM Detect kit is particularly useful for the analysis ofchallenging samples with incomplete profiles generatedwith GlobalFiler/NGM
• NGM Detect kit as a supplemental kit – dualamplification strategy
Summary – main advantages of NGM-Detect
Thank you for attention!
Acknowledge
mgr Agnieszka Parys-Proszekmgr Maria Wróbeldr Tomasz Kupiec
When used for purposes other than Human Identification or Paternity Testing the instruments and software modules cited are forResearch Use Only. Not for use in diagnostic procedures.Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo FisherScientific products presented by third parties during this seminar.Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, includingregarding intellectual property rights and reported results.Parties presenting images, text and material represent they have the rights
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