development of the ampfℓstr ngm select pcr amplification kit

1 | © Life Technologies 2011 | 4/5/2011

Development of the AmpFℓSTR®

NGM SElect™

PCR Amplification Kit:

Discussions of Validation, Sequence Variation, and Concordance


Presentation Outline

• AmpFℓSTR®

NGM SElect™

PCR Amplification Kit−

Kit Overview

• Developmental Validation Results−

Sensitivity Study−

Inhibition and Degradation Studies−

Species Specificity−

Precision Study−

Population Study>

Concordance>

Allelic Size Distribution>

Stutter Evaluation−

Mixture Study−

Annealing Temperature Study

• Design and Implementation of SE33 Primer Sequences−

Additional investigations into discordant genotypes


• Robust, single amplification STR multiplex kits optimized for use with database and challenging casework samples

• Developed specifically in accordance with the requirements for new multiplexes stipulated by the ENDAP/ENFSI groups for use in laboratories that will utilize the expanded European Standard Set of loci

• Combine

the latest advances

in primer synthesis, buffer optimization and thermal cycling optimization to deliver high sensitivity and

improved STR performance for forensic and database samples in one easy workflow


AmpFSTR®

NGM SElect™

Kit

Kit

Configuration

SE33 is included in the NGM SElect™

Kit using primers redesigned from the SEfiler

Plus™

Kit

New SE33 amplicon range 311-446 bp

Overall kit amplicon size range 70-446 bp


AmpFSTR®

NGM SElect™

Kit

Allelic Ladder

The NGM™

and NGM SElect™

Kit allelic ladders genotype the same range of alleles for loci common to the SGM Plus®, Identifiler®

and SEfiler

Plus™

kits

Multiple microvariant

virtual bins are included in the SE33 bin set for the NGM SElect™

kit to facilitate efficient genotyping and ensure concordant genotypes with the SEfiler

Plus™

kit


AmpFSTR®

NGM SElect™

Kit

Kit

Components & Thermal Cycling Conditions

The NGM SElect™

kit is being validated using the GS600 v2 size standard for maximised data precision and compatibility with the 3500 Series Genetic Analyzers

The NGM SElect™

kit provides the option of a 29 or 30 cycle

amplification protocol to maximise laboratory flexibility


AmpFSTR®

NGM SElect™

Kit

Optimised

Injection Recipe

PCR Product/Allelic Ladder

1.0 µLGS600 v2 Size Standard

0.5 µLFormamide

9.5 µL_______________________________Total

11 µL

Formamide volume increased by 1 µL to maximise denaturation

capacity for large multiplexes


Developmental Validation of the AmpFℓSTR®

NGM SElect™

Kit


NGM SElect™

Kit Developmental Validation

Experimental OutlineTest Kit/Instrument Test

Sensitivity StudyNGM SElect™

Kit (29 & 30 cycles)NGM™

Kit (29 & 30 cycles)SEfiler

Plus™

Kit (30 cycles)

2 control DNA samples:1 ng

to 31 pg

Degraded DNA Raji

DNA degraded with DNase

I

Inhibition Study Control DNA 007 with hematin

and humic

acid inhibitors

Population Study NGM SElect™

Kit (29 cycles) 1,276 individual human genomic DNA samples from four ethnic groups

Instrument Concordance 3130xl, 3130, 3100, 3500xL 42 genomic DNA samples from population sample set

Species Specificity NGM SElect™

Kit (29 cycles) 15 non-human genomic DNAs

Inheritance Study NGM SElect™

Kit (29 cycles) Three CEPH families each with 13-15 individual DNA samples

Mixture Study NGM SElect™

Kit (29 cycles) Series of mixtures with different ratios of 2 DNA samples

Allelic ladder sizing precision NGM SElect™

Kit 5 x 16 Allelic ladder Injections 3130xl

Master Mix Guard Band Studies NGM SElect™

kit (29 cycles) 3 x Control DNA Samples

Thermal Cycling Guard Band Studies NGM SElect™

kit (29 cycles) 3 x Control DNA Samples


Sensitivity Study Example Profiles1 ng

007 Control DNA 125 pg 007 Control DNA

3000 500


Sensitivity Study: Comparison of Mean Peak Heights

Mea

n Pe

ak H

eigh

t (R

FU)

NGM SElect™

Kit (29 Cycles)

NGM™

Kit (29 Cycles)

NGM SElect™

Kit (30 Cycles)

NGM™

Kit (30 Cycles)

SEfiler

Plus™

Kit (30 Cycles)

Input DNA Concentration


Degraded DNA Study Example Profiles: NGM SElect™

Kit, 29 Cycles

Non-degraded

3 U

4 U

5 U

6 U


Degraded DNA Study Example Profiles: SEfiler

Plus™

Kit, 30 Cycles

Non-degraded

3 U

4 U

5 U

6 U


Recovery of Information from Degraded Samples

6 U DNase

I

MiniFiler™ Kit

MiniFiler™

KitNGM™

Kit

Data courtesy of Instituto Nacional de Toxicología y Ciencias Forenses. Servicio de Biología, Madrid


Hematin

Inhibition StudyNGM SElect™

Kit, 29 Cycles SEfiler

Plus™

Kit, 30 Cycles

No Inhibitor

50 µM

150 µM

300 µM


AmpFSTR®

NGM SElect™

Kit

Species Specificity

Dog

Cat

Horse

Pig

Bovine

Sheep

Mouse

Rat

Rabbit

Hamster

Chicken

Bacterial Pool


AmpFSTR®

NGM SElect™

Kit

Species Specificity –

Higher Primates

Chimpanzee

Gorilla

Macaque


Population Study: Genotype Concordance

• Effect of Primer Binding Site Mutations on Genotype Concordance −

1Allelic dropout in 5 x NGM™

kit amplifications at D22S1045−


kit amplifications at Amelogenin−


kit amplifications at D2S441−

4Allelic dropout in 1 x SEfiler

Plus™

kit amplification

at SE33

AmpFℓSTR®

Kit

Ethnic Group

African-American(344)

Caucasian(346)

Hispanic(390)

Korean(1963)

NGM™

Kit 98.5%1 99.4%2 100% 95.4%3

NGM™

Kit (New) 100% 100% 100% 100%

SEfiler

Plus™

Kit 99.7%4 100% 100% 100%

NGM SElect™

Kit v NGM™

& SEfiler

Plus™

Kits


Population Study: Effect of SNP-Specific Primers

Sample ID Ethnicity Affected Locus

Genotype NGM™

KitGenotype

NGM SElect™ Kit

IB 0031 AA

D22S1045

14 14,15

IB 0133 AA 16 15,16

IB 0164 AA 17 15,17

IB 0169 AA 12 12,15

IB 0285 AA 8 8,15

BB 1054 Korean

D2S441

12 9.1,12

BB1056 Korean 10 9.1,10

BB1081 Korean 14 9.1,14

BB 1085 Korean 11 9.1,11

BB 1091 Korean 12 9.1,12

BB 1212 Korean 10 9.1,10

BB 1213 Korean 11 9.1,11

BB 1232 Korean 12 9.1,12

HC 1022 Korean 11.3 9.1,11.3

IB 0423 CaucasianAmelogenin

Y X,Y

IB 0532 Caucasian Y X,Y

Inclusion of 3 SNP-specific

primers permits recovery of null

alleles at the D22S1045,

D2S441 and Amelogenin loci


Population Study: Allelic Size Distribution of 42 Samples Relative to the Allelic Ladder

The sizing differential of sample alleles relative to allelic ladder alleles must be within ±

0.5 bp

to ensure accurate

genotyping


Population Study: Stutter Calculations

Stutter percentages are calculated from population study data run at 29 cycles of amplification


GeneMapper®

ID & ID-X Stutter Filters

• Stutter filters are calculated as [mean observed stutter + 3SD]

• Stutter filters contained in Beta release analysis files have been updated based on the outcome of developmental validation studies

• The -4, -3 and +3 bp

stutters are included as stutter in filters in NGMSElect_panel_v1 and in NGMSElect_stutter_v1X

• The -2 bp

stutter filters are not included NGMSElect_panel

v1 due to functional limitations of the GeneMapper®

ID Software

Locus % Stutter Filter Beta Release

% Stutter Filter Final Release

D10S1248 12.89 12.04

D12S391 15.84 14.77

D16S539 10.57 9.87

D18S51 13.89 13.81

D19S433 11.06 11.02

D1S1656 14.16 13.60

D1S1656 (-2nt) N/A 7.51

D21S11 11.40 10.43

D22S1045 17.99 17.14

D22S1045 (+3nt) 7.10 6.65

D2S1338 13.55 12.32

D2S441 9.47 9.03

D3S1358 13.77 11.91

D8S1179 10.82 9.93

FGA 12.61 11.52

SE33 13.52 15.12

SE33 (-2 nt) 5.66 5.95

TH01 5.26 4.93

vWA 11.82 11.18


Examples of NGM SElect™

Kit Stutter Filters

• -2 bp

stutters were observed at the D1S1656 and SE33 loci during developmental validation population studies−

Arise due to sequence motif complexity−

Detectable above the 50RFU threshold at sufficient frequency to warrant inclusion of a stutter filter (29 cycles)

• +3 bp

stutter observed frequently at the D22S1045 locus−

Trinucleotide

loci generally yield a higher percent stutter than longer repeat unit loci−

Detection of plus stutter at other loci is more likely with Next

Generation STR kits due to the nature of the optimised buffer systems

−

Detection of plus stutter is more likely at higher cycle numbers

(e.g. 30 cycles)


Stutter Calculations Considerations

• Forensic laboratories should evaluate the appropriate stutter filter settings for their operations as part of internal validation studies

• Stutter filter values included in GeneMapper®

ID and GeneMapper®

ID-X Software are calculated from populations studies conducted as part of each developmental validation project−

All amplifications ~ 1 ng

input DNA−

Wide range of alleles evaluated (>1000 samples)−

Filter value = Mean Stutter + 3SD

• Low concentration samples may yield higher than expected stutter

peaks


Mixture Study

1:0 (Major)

15:1

10:1

7:1

3:1

1:1

0:1 (Minor)

Full profile for the minor contributor obtained at 7:1 ratio (125 pg)


Annealing Temperature Study

57oC

58oC

59oC

60oC

61oC


AmpFℓSTR®

NGM SElect™

Kit

Additional Analysis Considerations


Rare Alleles at Locus Extremes Overlap of D3S1358 and D1S1656 Alleles

Rare D1S1656 Allele 7

Labelled as D3S1358 Allele 20


Assignment of Rare Alleles Use of Peak Height and Consideration of Allele Frequency

a b c d

D3S1358 D1S1656 D3S1358 D1S1656

a b c

D3S1358 D1S1656

a b c

D3S1358 D1S1656

a b c

ab,bcab,cc

ab,cd aa,bc

Other scenarios are theoretically possible but statistically highly unlikely


Assignment of Rare Alleles Use of GeneMapper®

ID-X Software

Single allele displayed: 12

Green marker header bar

No flags fired

D1S1656 Display


Assignment of Rare Alleles Use of GeneMapper®

ID-X Software

3 alleles displayed: 15,16,20

Red marker header bar

AN & PHR flags fired

D3S1358 Display


Assignment of Rare Alleles Use of Overlapping Marker Ranges in GeneMapper®

ID-X Software

2 alleles for D1S1656: OL,12

3 alleles for D3S1358: 15,16,20

Red header bars for both loci

OVL flag fires for both loci


Design & Implementation of SE33 Primer Sequences


Diagnosis & Characterisation of Sequence Variants at the SE33 Locus

• Population studies conducted by Life Technologies revealed 19 discordant SE33 results between SEfiler

Plus™

kit and prototype SE33 primers for the NGM SElect™

kit

• Discordance caused by a difference in allele size of approximately 1 bp, reproducible on different instrument platforms

• Discordance also visible in genotypes generated by other commercially-available kits

• Forensic laboratories have reported the discordance and sent samples to Life Technologies for genotype confirmation and sequence analysis

• Comprehensive sequence analysis has been conducted to understand

the cause of the discordance and the mechanism by which it occurs


Examples of Discordance at the SE33 locus between Prototype SE33

Primers and the SEfiler

Plus™

KitSample IBB 297 Sample IBB 298 Sample IBB 509

(20.2, 21)

(20.3, 21)

(16, 18)

(16,18.1)

(26.2, 30.2)

(26.3, 30.2)

A total of 19 samples exhibited the shift, predominantly from African American origin

Prototype SE33

Primers

SEfiler

Plus™

Kit


Discordant SE33 Genotypes Between Different Primer Sets: Sample IBB 509

(26.3, 30.2) (26.2, 30.2)

(26.3, 30.2) (Null, 30.2)PowerPlex®

ESX 17PowerPlex®

ESI 17

Prototype SE33 Primers NGM SElect™

Kit

SEfiler

Plus™

Kit SE33 Genotype = 26.2, 30.2


SE33 Discordance Investigations Sequence Analysis Results: Life Technologies Samples

Prototype SE33 Primers

SEfiler

Plus™

Kit Primers

SNP-Containing Region

CG(C/T)(G/A)GAGACC(G/A)CG

Type I Type II Type III

Analysis of 19 samples revealed three distinct single nucleotide polymorphisms

None of these mutations cause a change in length of the amplicon, genotype discordance originates from a mobility shift


NIST Analysis of Mobility Shift Samples ESI 17 v NGM SElect™

Kit Results

NGM SElect™

Kit

ESI 17

(18,24.2)

(18,24.3)

(21,22.2)

(21,22.3)

(20,21.2)

(20,21.3)

(23.2,28.2)

(23.3,28.2)

(13.2,19)

(13.3,19)


LKA Mecklenburg-Vorpommern

Analysis of Discordant Samples

SEfiler

Plus™

Kit v ESI 17 Results

SEfiler

Plus™

Kit

ESI 17

(14.2,16)

(14.3,16)

(14.2,18)

(14.3,18)

Mobility shift observed in 2 samples (Father and Daughter)


Life Technologies Analysis of LKA Mecklenburg-Vorpommern

Samples: NGM SElect™

Kit, ESI 17 and ESX 17 Results

ESI 17 ESX 17NGM SElect™

Kit

(14.2,16)(14.3,16)(14.2,16)

Sample 002Daughter

Sample 003Father

(14.2,18)(14.3,18)(14.2,18)


SE33 Mobility Shift Investigations Sequence Analysis Results: LKA Mecklenburg-Vorpommern

Wild Type Allele Mutant Allele

All mutant alleles showed the same G-A transition


NGM SElect™

Kit SE33 Primers

Restoration of Concordant GenotypesIBB 115 IBB 297 IBB 298 IBB 509

More than 80 different primers for the SE33 locus were investigated to identify the final, optimum configuration

Prototype SE33

Primers

NGM SElect™

Kit


Mobility Shift & Sequence Investigations Summary• 1 bp

mobility shift observed in SE33 amplicons

from 19 Life Technologies samples and confirmed in 2 samples provided by a customer laboratory

• Mobility shift present in SE33 amplicons

produced using NGM SElect™

kit prototype SE33 primers and the ESI 17 kit

• Mobility shift shown in all cases to result from sequence variants as opposed to the more commonly encountered length variants

SE33 Repeat Region

SNP Region

gDNA

SEfiler

Plus™

Kit Forward Primer

Reverse Primer

SEfiler

Plus™

Kit

Reverse Primer

Prototype SE33

• 3 types of SNPs

have been identified to date−

SNP types I, II and III all cause the mobility shift

−

SNP types II and III also cause dropout of SE33 alleles in the ESX 17 kit

• Final NGM SElect™

kit primers exclude the SNP region from the amplicon and remove

the risk of the mobility shift occurring


AmpFSTR®

NGM SElect™

PCR Amplification Kit

• Results of a comprehensive developmental validation study demonstrate that the NGM SElect™

kit is a robust and reliable multiplex, suitable for use on all

types of forensic samples

• The molecular structure of the SE33 locus posed significant challenges during the development of new primers for use in the NGM SElect™

kit

• The new SE33 primer sequences have been designed to maximise concordance with genotypes generated by the SEfiler

Plus™

kit

• Primer development investigations highlighted a SNP-containing region which, if contained within the SE33 amplicon, causes a mobility shift and subsequent incorrect allele designation in affected samples

• The NGM SElect™

kit also includes three additional primers designed to address primer binding site mutations identified at the Amelogenin, D2S441 and D22S1045 loci−

A project is underway to introduce these primers into the existing NGM™

kit to maximise concordance between the two systems


Thank You!

©

2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. PowerPlex

is a trademark of Promega Corporation.

For Research, Forensic or Paternity Use Only. Not intended for any animal or human therapeutic or diagnostic use

development of the ampfℓstr ngm select pcr amplification kit

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