results of the validation study of the applied biosystems ......profile number of additional alleles...

Vienna, 17 May 2017r Tomasz Kupiec

Institute of Forensic Research, Kraków,Poland

Results of the validation study of the Applied Biosystems™ NGM Detect™ kit

Workflow Magnetic extraction

Organic extraction (bones)

Applied Biosystems™ Quantifiler™ Trio

Applied Biosystems™ NGMDetect™ NGMSelect™ GlobalFiler™ Yfiler™ Plus

Electrophoresis Applied Biosystems™ 3500xL Genetic Analyzer

X2

Main characteristics

Sample input volume: NGMDetect: 15µl 500pg NGMSelect: 10µl 1000 pg GlobalFiler: 15µl 1000 pg

16 autosomal STRs, Amelogenin, Yindel 7 mini-STRs below 230 bp Internal quality control system

D12S391 primers – share with GlobalFiler and NGMSelect kits, Double typing procedure – complementary marker positions

IQC, Yindel

IQC system - two synthetic DNA fragment: LMW (IQCS) and HMW (IQCL) - quality control of PCR

SGM, Identifiler

Identifiler Plus, NGM, NGMSelect

GlobalFiler

NGMDetect

Amplification time

Validation experiments

- Sensitivity, - Mixture study, - Stability – inhibitors, - Casework samples analysis – bones samples,

Threshold set-up: LOD, LOQ, Stochastic threshold, Stutter ratio,

Procedure accreditation according to EN 17025:2005

Sensitivity – 500 pg

- Three replicates of control DNA 007 serial dilutions

Sensitivity - 250 pg

Sensitivity – 125 pg

Sensitivity - 63 pg

Sensitivity - 31 pg

Sensitivity - stochastic effect (31 pg) Sensitivity - stochastic effect (31 pg)

Sensitivity - 16 pg

Sensitivity - 7 pg

Sensitivity - summary

PHR > 60%

No results

PHR < 60%

NGM Detect Inj. 20”

NGM Detect Inj. 10”

LOQ 600 (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

Thresholds:

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 DNA XY 007

Control DNA X 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 analysis Mixture analysis Three replicates

Mixture analysis

1:1

Mixture analysis Mixture analysis

Mixture analysis

Mixture analysis

STR n-4 n-2 n+4 n-3 n+3 D2S1338 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 0,0919 D10S1248 0,1588 - - - -

Stutter:

Casework samples analysis – bone samples 17 bone 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 samples

GlobalFiler

NGM Detect

GlobalFiler

Casework samples analysis – tooth DI=8

D16S539, SE33, D2S1338 D2S441 D2S441 D16S539, SE33, D2S1338 D2S441 D16S539, SE33, D2S1338 D16S539, SE33, D2S1338 D2S441 D16S539, SE33, D2S1338

NGM Detect


D16S539, SE33, D2S1338 D2S441 D16S539, SE33, D2S1338 D2S441 D16S539, SE33, D2S1338 D2S441 D16S539, SE33, D2S1338 D2S441

•  NGM Detect = 11 STRs

•  GlobalFiler = 13 STRs

•  NGM Detect & GlobalFiler = 18 STRs

NN & mother LR=263

LR=85

LR=12135


GlobalFiler

Casework samples analysis – petrous bone DI=19

D2S441 D16S539, SE33, D2S1338

NGM Detect


D16S539, SE33, D2S1338 D2S441

•  NGM Detect = 11 STRs

•  GlobalFiler = 14 STRs

•  NGM Detect & GlobalFiler = 18 STRs


Humic acid inhibition- Quantifiler Trio

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

HA 0 HA 50 HA 100 HA 200 HA 400 HA 600 HA 800 HA 1000 HA neg

Humic acid concentration in final volume of qPCR mixture [ng/ul]

DN

A q

uan

tity

[n

g/u

l]

26

26,5

27

27,5

28

28,5

29

29,5

30

30,5

IPC

Ct v

alue

largesmallIPC

1 2 3 1- 1000ng/ul of HA in NGM Detect PCR 2- 1500ng/ul of HA in NGM Detect PCR

3- 2500ng/ul of HA in NGM Detect PCR


Inhibition

•  Piece of sterile cotton fabric (5cm x10 cm) was attached close to the t-shirt label (neck) and worn for 8h by volunteers.

•  Fabric was stored for 5 days in sterile paper envelope in a drawer (standard storage conditions, 25ºC, dry environment),

•  Direct PCR of the fiber from the fabric,

Direct PCR

Person Number of fibers

Number of alleles detected (LOQ)

Complete profile

Number of additional alleles

A

1x1,0cm 1

32

0

2x1,0cm 32 1

3x1,0cm 31 1

B

1x1,0cm 12

32

0

2x1,0cm 34 2

3x1,0cm 30 3

Comparison of DNA profiles generated from fibers via direct PCR, using NGM Detect

Direct PCR

Person A – 2x1,0cm of cotton fiber

Direct PCR

Person A – 3x1,0cm of cotton fiber

Direct PCR

Person B – 2x1,0cm of cotton fiber

Direct PCR

Person B – 3x1,0cm of cotton fiber

Direct PCR

•  NGM Detect is particularly useful for the analysis of challenging samples: resistance to inhibition, high sensitivity,

•  NGM Detect kit provide complementary marker positions to NGMSElect and GlobalFiler kits,

•  Quality control markers (IQCS, IQCL) provide a good control of amplification process,

•  Direct amplification of contact traces is possible.

Summary

•  Agnieszka Parys-Proszek •  Magdalena Marcińska •  Maria Wróbel •  Andrzej Doniec •  Miłosz Januła

Thermo Fisher SCIENTIFIC For Forensic or Paternity Use Only. When used for purposes other than Human Identification the instruments and software modules cited are for Research Use Only. Not for use in diagnostic procedures. Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration

Acknowledgments

Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration When used for purposes other than Human Identification or Paternity Testing the instruments and software modules cited are for Research 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 Fisher Scientific 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, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.

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results of the validation study of the applied biosystems ......profile number of additional alleles...

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