validation of a single tube fluorescent multiplex assay

Indian Journal of Biotechnology

Vol 7, January 2008, pp 41-49

Validation of a single tube fluorescent multiplex assay for simultaneous typing of

20 Y-STR loci

Sanghamitra Sahoo1, 2

and V K Kashyap1, 2*

1National DNA Analysis Centre, Central Forensic Science Laboratory, 30 Gorachand Road, Kolkata, India

Received 12 June 2006; revised 27 April 2007; accepted 23 July 2007

This paper describes a single tube assay for 20 fluorescent labelled Y-STR loci, which is rapid and robust state-of-art

multiplex system. Reaction conditions, including annealing temperature, concentration of primers, magnesium and template

DNA, DNA polymerase and reaction volume, were optimised to yield robust amplification. The assay could withstand

moderate fluctuations in reaction parameters with no affect on haplotype analysis. Sensitivity and robustness of the

multiplex system is revealed from the examination of different matrices and environmental conditions wherein complete

haplotypes could be obtained in all the samples with no failures except those exposed to severe environmental stresses.

Genotyping of the 20 Y-STR loci showed that all loci included in the multiplex are highly polymorphic in the Indian

populations and its inclusion increases the discriminatory power of the marker system. It could be summarized that the

developed Y-STR multiplex assay is a simple, sensitive and robust system highly suitable for concurrent analysis of 20

polymorphic Y-STR loci.

Keywords: multiplex PCR, Y-STR, DNA profiling, validation

Introduction Multiplex polymerase chain reaction (PCR) is a

system in which two or more loci are simultaneously

amplified in the same reaction. This method has been

successfully applied in many areas of DNA typing,

including analyses of deletions, mutations1,2

and

polymorphisms at short tandem repeat (STR) loci3-8

since its first report9. STRs or microsatellites contain

2-6 bp repeat units which are tandemly arranged and

widely dispersed throughout the eukaryotic genome.

Analysis of these tandemly repeated DNA sequences

is of fundamental importance not only in population

biology, such as human identification, understanding

origin of man, migratory histories of populations,

phylogenetic distribution of various plant and animal

species, and other evolutionary processes, but also in

linkage analysis of genetic traits. Multiplex STR

analysis, therefore, has emerged as an indispensable

assay as it requires only sub-nanogram amounts of

DNA, is less labour intensive, provides easy

interpretation of data, high discriminatory power and

reliable results. Although a large number of

multiplexed commercial kits are available for human

autosomal STR markers [Powerplex® 2.1, Powerplex

®

16 system (Promega Corp. Madison, WI), ABI

AmpFl STR® Profiler

™, COfiler

™ and Identifiler

™)],

the same is not the state for the microsatellites of Y-

chromosome. The reason for this lacuna is because

until recent past, the number of STRs known on the Y

chromosome was limited10-14

. In addition, lack of

suitable Y-STR mutiplex system is also another

reason why the Y-chromosome has not been as

extensively used as the autosomal STRs in human

identification and population genetic studies.

Recently, however, three commercial Y-STR

multiplex systems: Y-PLEXTM

6 kit (ReliaGene

Technologies Inc.), Powerplex Y System (Promega

Corp.) and AmpFLSTR Y filer kit (Applied

Biosytems) are available; these include only 6, 12 and

17 Y-STR loci, respectively15,16

. Although the Y

chromosome harbors diversity which is only 20% as

that of the autosomes17

, a large number of Y-STRs

have been identified in recent years, and a few of

them have also been used for human identification,

particularly paternity testing, and evolutionary,

genealogical and anthropological studies18,19

.

_________________

*Author for correspondence:

Tel: 91-120-2400027; Fax: 91-120-2403014

E-mail address: [email protected]

Present Address: 2National Institute of Biologicals, A-32, Sector 62,

Institutional Area, Noida 201 307, India

INDIAN J BIOTECHNOL, JANUARY 2008

42

Majority of the STRs on the Y-chromosome are

located in the non-recombining region and thus are

passed down the generations without recombination.

In addition, because the Y-chromosome is present in a

haploid state, it is exceptional and unparalleled

compared with any of the known markers, for tracing

male movements either of the present-day or events of

the past20

. However, lack of recombination also

suggests that individual markers cannot be combined

by the product rule and to achieve power of

discrimination similar to those of autosomal STR

markers21,22

, a large number of Y-STR loci should be

included in the analysis. The Y-STR markers find

application in a wide range of forensic investigations,

especially in cases where father of the male child is

deceased17,23

, identification of human remains in mass

disasters24

and offenders, particularly in sexual assault

cases25

, where the male component needs to be

identified from the mixed male and female fractions

of the body fluids26

and where methods such as

differential lysis is unsuccessful, as in azoospermic

semen samples. In addition, these STRs are equally

important in evolutionary biology, archeological and

anthropological studies pertaining to male

population17,18,27-29

. Few studies, using Y-STRs, were

primarily based either on monoplexes or multiplexes

carried out as 2-3 tube reactions. Since these are

laborious and time-consuming protocols, single tube

multiplex methods are much in demand30

. The

development of a large multiplex will lead to newer

possibilities for studying which Y-STRs have the

most significant impact on a particular population by

enabling more rapid collection of the data from

population samples. Understanding the genetic

structure of individual populations will help in

maintaining national DNA databases, which is

increasingly essential31-33

for assessing the strength of

match evidence in DNA profiling cases. Therefore,

development of a Y-STR multiplex system is not only

of immense value in justice delivery system34

but also

in understanding the Indian scenario of male

evolutionary and population genetics. We have,

therefore, attempted to develop, standardize and

validate a set of 20 Y-STR loci for a single tube

multiplex reaction that will provide a sensitive,

reproducible, robust and rapid PCR assay.

Materials and Methods Reagents for PCR

10X PCR gold buffer containing 500 mM KCl, 150

mM Tris-HCl, pH 8.3, 25 mM MgCl2 and AmpliTaq

Gold®

DNA polymerase were procured from Applied

Biosystems, Foster City, USA. The nucleotides

(dNTP- 100 mM each) and glycerol was purchased

from Promega Corp. (Madison, WI, USA) and SRL

(Mumbai, India), respectively. Customised

fluorescent labelled and non-fluorescent primers were

procured from Isogen Bioscience BV, The

Netherlands and reconstituted in TE buffer to a final

concentration of 1 nmol/µL each. The working stocks

of all primers were prepared in milli-Q water for a

concentration of 20 pmol/µL.

Selection and Description of Y-STR loci

Our previous standardization experiments included

eight Y-STR loci35

, which constituted the minimal

haplotype required for human identification

recommended by the ISFG guidelines (protocol

unpublished). The “minimal haplotype” includes

tetranucleotide Y-STR markers DYS19, DYS385a/b,

DYS389I/II, DYS390, DYS391, DYS393 and a

trinucleotide, DYS392. However, with the availability

of information on polymorphism for new Y-STR

loci10, 36-40

, we considered their inclusion as an asset in

enhancing the power of discrimination manifold. The

loci incorporated in the new multiplex include one

dinucleotide locus YCAIIa/b, two other trinucleotide

loci DYS388 and DYS426, and four tetranucleotide

repeat loci DYS460, H4, DYS437 and DYS439. In

addition to these, there are two pentanucleotide repeat

loci DYS438 and DYS447 and one hexa repeat

nucleotide locus DYS448. Three primer pairs,

DYS389, DYS385 and YCAII generate two

amplicons each, resulting in a total of 20 Y-STRs.

The detailed information on loci used in the 20-Y-

STR multiplex is given in Table 1. We carried out

standardization and in-house validation of 20 Y-STR

loci in a single tube multiplex reaction by using these

primers described previously41

. The multiplex system

would also help in examining whether the Indian

populations harbour similar level of polymorphism in

all the selected Y-STR markers as those described by

the European populations so that a uniform set of

markers could be easily used in routine forensic

investigations to provide a high power of

discrimination.

Description of Primers

The amplification of the selected loci was

carried out using NIST primer sequences

obtained from the Y-STR database site

(http://www.cstl.nist.gov/biotech/strbase/). Although

SAHOO & KASHYAP: SINGLE TUBE FLUORESCENT MULTIPLEX ASSAY FOR SIMULTANEOUS TYPING OF 20

43

the genetic recombination between X and Y

chromosome is low, certain degree of homology exists

between the two chromosomes because of their common

evolutionary history42

. Therefore, the objective was to

incorporate those primers which have enough specificity

to ensure preferential amplification of Y-chromosome

sequences and minimize or eliminate any interference

from X chromosome. The primers were aligned with

each other and also with the sequence database at the

National Center for Biotechnology Information (NCBI)

using the Basic Local Alignment Search Tool (BLAST)

program to rule out any possibility of dimerization and

to determine the extent of homology with the X

chromosome or to any other chromosome. The primers

are labelled with four different fluorescent dyes, 6′-FAM

(blue), VIC (green), NED (yellow) and PET (red), where

different flurochrome would detect overlapping

fragment sizes.

Test Samples

Genomic DNA was obtained from blood or tissue

samples from both human and non-human sources

following organic extraction protocol43

.

Basic PCR Protocol

The basic PCR (20 µL reaction volume) included:

autoclaved milli-Q water, 1X PCR gold buffer, 1.5 mM

MgCl2, 200 µM each dNTP, 1U AmpliTaq GoldTM

, 5%

v/v glycerol (if used), primer (0.2-1.2 µM each) and

genomic DNA. The amplicons were checked by

electrophoresis on Nusieve®

Agarose gels (FMC

Bioproducts, Rockland, ME, USA) containing

Ethidium bromide in 1X TBE [0.09 M Tris-Borate;

0.002 M EDTA (pH 8.0)] buffer and detected under the

UV transilluminator. All the amplification reactions

included a known DNA sample as male positive

control, a female DNA as negative control and an

amplification negative control.

The basic PCR protocol included (1) Amplification

of each loci as monoplexes and (2) Multiplexing of the

Y-STR loci through a series of standardization steps:

i) Standardization of annealing temperature

ii) Optimization of primer concentrations

iii) Optimization of DNA polymerase and magnes-

ium concentrations

iv) Validation of multiplex assay

Results and Discussion Single Locus PCR

Each of the primer pairs was amplified individually

at the same annealing temperature (55ºC) in 1x

reaction condition with 0.5 µM primer concentration

and without glycerol (Table 2). A high primer

concentration was maintained for checking the

efficacy of primers under similar amplification

conditions. Fig. 1 shows the agarose gel image for

successful amplification of all the single Y-STR loci.

Multiplex PCR

Combining the primers together for amplifying

many loci simultaneously requires

alteration/optimization of certain parameters of the

reaction44

. Initially, all the primers were added in

equimolar amounts in various combinations under

single locus PCR conditions in addition to 5%v/v

glycerol, to test the individual primer concentrations

and other parameters for a successful multiplex

reaction. For the initial standardization, all primers

were mixed in 0.5 µM concentrations, of which only

8.0 µL of Y-STR primer mix was added to the 20 µL

reaction mix. Fig. 2 shows the 4% agarose gel

photograph for positive amplification with Y-STR

multiplex. The final primer concentrations for each of

the primer pairs are given in Table 3. Performance of

the PCR-based STR assays can be influenced by

numerous factors, including the difference in

Table 1—General information on loci used in the 20 Y-STR

multiplex system

Marker Name Repeat motif Gen Bank

accession

Reference

allele

DYS19 TAGA AC017019 (r&c) 15

DYS385 a/b GAAA AC022486 (r&c) 11

DYS389 I (TCTG) (TCTA) AC004617 (r&c) 12

DYS389 II (TCTG) (TCTA) 29

DYS390 (TCTA) (TCTG) AC011289 24

DYS391 TCTA AC011302 11

DYS392 TAT AC011745 (r&c) 13

DYS393 AGAT AC006152 12

YCAII A/B CA AC015978 23

DYS388 ATT AC004810 12

DYS426 GTT AC007034 12

DYS437 TCTA AC002992 16

DYS438 TTTTC AC002531 10

DYS439 AGAT AC002992 13

DYS447

(TAATA)

(TAAAA) AC005820 23

DYS448 AGAGAT AC025227 22

DYS460

(A7.1) ATAG AC009235 (r&c) 10

Y-GATA-H4 TAGA AC011751 (r&c) 12

Reference allele refers to the number of repeats found in the

GenBank sequence, which must sometimes be made reverse and

complement (r&c) in order to maintain consistency with previously

used repeat motifs (42,46).


44

instrumentation, variability in quality and quantity of

DNA template being amplified, variations in reaction

and analysis conditions. Therefore, further

standardization of the multiplex was carried out after a

few preliminary rounds of detection of the amplicons

in a genetic analyzer for a high throughput format to

obtain accurate and precise size calling of the alleles.

Detection and Analysis of PCR Products

Detection of the amplicons was achieved with ABI

Prism® 3100 Genetic Analyzer 16-capillary array

system (Applied Biosystems) using the G5 matrix

filter for the 5-dye chemistry. GS500 Liz™ (Applied

Biosystems), which produced orange colour

fluorescence, was used as size standard for sizing

length of the amplicons in basepairs. Samples were

prepared in Hi-Di™ formamide (Applied Biosystems)

with GS500 Liz™ size standard (mixed in 25:1 ratio)

and 1 µL of PCR product. Electrophoresis were

performed at 15 kV for 44 min with a run temperature

of 60ºC using 3100 POP™-4 (Applied Biosystems),

1X Genetic Analyzer Buffer with EDTA and a 36 cm

array. Following data collection, samples were

analysed with Genescan® 3.7 (Windows NT, Applied

Biosystems) for sizing of the products. Allele

designations were made based on sizing bin windows

of up to ±0.15 bp.

Optimization of Reaction Conditions

Depending on the difference in peak heights of the

amplicons, appropriate adjustments were made in the

concentration of the critical reagents and

thermocycling conditions including reaction volume,

template and primer concentrations, annealing

temperature to obtain more balanced signal intensity.

Table 2—Cycling conditions for single locus and multiplex PCR

Single locus PCR Multiplex PCR

Pre-denaturation 95ºC, 11 min 95ºC, 11 min

Denaturation 94ºC, 1 min 94ºC, 1 min

Annealing 55ºC, 1 min 57ºC, 1 min

Extension 72ºC, 1 min 72ºC, 1 min

30 cycles 28 cycles

Final extension 60ºC, 45 min 60ºC, 45 min

Hold 4ºC 4ºC

Fig. 1—2% Agarose gel image of single locus PCR amplification

of 20 Y-STR using 1X buffer conditions at an annealing

temperature of 55ºC.

Fig. 2—Multiplex PCR amplification of 20Y-STRs at 57ºC using

the PE 9700 thermal cycler in a 4% agarose gel.

Table 3—Locus information, size range42 and final primer conc of

20 Y-STR loci

STR locus Size range

(bp)

Fluorescence

label

Allele

range

Primer conc

(µM)

DYS19 233-269 NED 10-19 0.76

DYS385 242-306 VIC 7-23 0.46

DYS388 151-175 NED 10-18 1.5

DYS389I 143-175 6-FAM 9-17 1.5

DYS389II 263-295 6-FAM 26-34 -

DYS390 189-233 VIC 17-28 1.5

DYS391 93-121 6-FAM 7-14 0.46

DYS392 290-318 NED 6-16 1.5

DYS393 109-133 VIC 9-16 0.92

DYS426 92-98 VIC 10-12 0.46

DYS437 186-198 6-FAM 14-17 0.46

DYS438 300-335 6-FAM 6-13 0.76

DYS439 210-230 6-FAM 16-21 0.46

DYS447 187-242 PET 22-29 0.46

DYS448 299-335 PET 18-26 0.30

DYS460 101-121 NED 7-12 0.46

H4 122-142 NED 8-13 0.46

YCAII 135-161 VIC 11-24 0.46


45

Reaction Volume and Template Concentration

Change in the reaction volume can alter the

concentration of PCR reaction components. Varied

reaction volumes (5, 10, 12.5, 15, 20 and 25 µL) were

evaluated to determine the effects on amplification.

When the concentrations were kept uniform,

amplification was seen at >10 µL volumes. To test the

sensitivity of the multiplex system, a serial dilution of

the test samples was performed to quantify the optimal

concentration of DNA template required for a robust

multiplex amplification. Template concentration of 10

ng per reaction volume gave more than 100 RFU and

was found to be sufficient for robust amplification.

Larger loci drop-outs were observed at concentrations

less than 5 ng template DNA.

Primer Titrations

Concentrations of the primers play a crucial role in

the amplification efficacy of loci in a multiplex

reaction. Therefore, adjustments in the primer

concentrations are critical to produce equivalent signals

for each locus. Primer concentrations between 0.1 µM

and 1.5 µM with 28-30 cycles and template between 1

and 25 ng were tested for determining relative signal

balance between loci. When high primer concentrations

were used, smaller loci were preferentially amplified.

With the final primer concentrations as given in Table

3, peak heights decreased in smaller loci and increased

in larger loci, giving a relative balance of signal

intensity. Locus drop-out was seen at primer

concentrations less than 0.2 µM and 5 ng of DNA.

Final reaction volumes were adjusted to 10 µL to

which primer volume was further reduced to 2.0 µL of

primer mix.

Variation of Annealing Temperature

Changes in the annealing temperature can affect the

specificity and balance of the amplified loci. To

examine these effects, amplification reactions were

performed at various annealing temperatures (55ºC-

60ºC) with a minimum of 25 ng DNA template

concentration. Variations in annealing temperatures

can also cause cross-reactivity and allele drop-outs.

The most commonly observed loci to drop-out at

higher annealing temperature include DYS19,

DYS389II, DYS447 and DYS388. Locus drop-out and

additional artifacts were observed at temperatures

above 58ºC, whereas at lower annealing temperature an

increased yield of the smaller loci caused a locus-to-

locus imbalance. The final annealing temperature was

maintained at 57ºC (Table 2).

Optimization of DNA polymerase and Magnesium Concentrations

The DNA polymerase plays a key role in

determining the proportion of amplification that could

be achieved. Therefore, variations in the

concentrations of AmpliTaq® Gold DNA polymerase

were evaluated. Between 1 and 5U/20 µL DNA

polymerase concentrations were examined with

template concentration of 1-25 ng and 28-30 cycling

reactions. Optimal balance of signal between loci

were seen at as low as 1U per reaction volume. No

locus drop-out was observed at this enzyme

concentration but the yield of larger loci was low.

Magnesium has significant effect in polymerase

activity and specificity, but EDTA in the samples can

inadvertently alter effective concentrations of

magnesium. Various titrations between 1-2.5 mM

were examined, where the optimal balance between

loci was seen at 1.5 mM magnesium concentration.

Locus-to-locus imbalance increased on decreasing the

concentration of magnesium to less than 1.2 mM.

Lower concentration resulted in locus drop-outs while

higher concentration led to the formation of too many

non-specific products.

Comparison of Thermal Cyclers

Different models of thermal cyclers have slightly

different heating and cooling properties. To evaluate

the consistency of yield across loci on different thermal

cyclers, two different instruments— Perkin-Elmer

GeneAmp® PCR System 2400 and 9700 were

examined. This exercise was carried out to rule out

biased haplotypes due to the usage of different PCR

machines. The signal intensity of each locus (RFU)

was compared to the total signal across all the loci for a

sample. No consistent performance differences were

observed between the thermal cyclers (Table 4).

Validation of the Multiplex

STR analysis of forensic samples in particular,

relies on two important parameters (i) specificity of

primers for humans and (ii) sensitivity for detection45

.

Thus, validation studies of the multiplex system were

carried out on different samples and matrices to verify

the species specificity, sensitivity, mixture analysis

and effect of environmental stresses. The specificity

of multiplexes for amplification was examined with

five different animal species, including rat, frog, fish,

chicken and bovine sp. None of the tested samples

yielded positive amplification. This assay with high

degree of human specificity is essential for accurate

result interpretation in case of contamination from

non-human sources. The sensitivity of the multiplex


46

system was tested by quantifying the optimal amount

of template DNA required for reliable amplification in

serially diluted DNA samples. An advantage of Y-

STR analysis even with low template concentration is

that while in the case of autosomal markers, allele

drop-out creates potential for misinterpretation at

heterozygous locus, drop-out of a Y-STR loci will

produce no information except in cases of male/male

mixtures or bi-allelic loci such as DYS385a/b and

YCAII a/b. Further, to demonstrate that the sampling

method does not produce biased haplotype profile for

the Y-STRs, reproducibility with liquid blood, dried

bloodstains, buccal swabs, semen sample and autopsy

tissue was carried out on various male samples. In all

instances, different samples produced complete,

correct and identical profiles. Exposure to different

environmental conditions, such as temperature and

sunlight exposure is known to affect the quality of

DNA resulting in degradation of larger templates and

decreased yield of larger amplicons. To evaluate this,

DNA was extracted from bloodstains exposed to

various natural and laboratory-controlled conditions

for different time periods. All the samples exposed to

50ºC or lower temperatures and a range of light

exposures produced complete haplotypes (Fig. 3). To

confirm the effect of PCR inhibitors on substrate

materials which can adversely affect the performance

of amplification, we carried out amplification and

generated complete profiles from various matrices

such as, gauze, wood, glass, and soil, etc. One of the

salient features of Y-STRs is its utility in analysis of

mixed samples. Male and female fractions in various

concentrations and combinations, from a ratio of 10:1

to 1:10 were analyzed. It was carried out to exclude

the probability of any female sample getting amplified

and secondly, to detect inhibitory concentrations of

female DNA for Y-specific amplifications. Large

concentrations of female fractions affect the

amplification of higher loci. Even with varying ratios

of the male samples in the male/male mixtures,

complete and correct haplotypes could be generated.

These data suggest that this multiplex has the level of

specificity and sensitivity to reliably produce results

for proportions to male/female mixtures generally

obtained in forensic samples. Determining the number

of donors in the male/male mixtures only becomes

difficult when bi-allelic loci are taken during

interpretation.

Population Studies

As a part of the validation study and to determine

the extent of polymorphism of the selected Y-STR

markers such that they are able to differentiate

between unrelated males with a high degree of

significance, we tested the multiplex system on

various population samples46-50

. We observed that a

Fig. 3—Validation of the 20-Y-STRs multiplex PCR in liquid blood and stain samples in guaze and Whatman paper.


47

total of 1240 distinct Y-STR halotypes in 1297

unrelated, healthy Indian males analysed with the 20

Y-STR multiplex, with complete profiles were

generated in more than 90% of the samples analysed.

The power of discrimination between individuals with

the 20 Y-STR was estimated to be 0.999. The number

of alleles found for each locus ranged from 7-18, with

the exception of DYS385 and YCAII loci, which gave

81 and 55 genotypes, respectively. All the Y-STR loci

depicted gene diversity values greater than 0.5, even

for those loci that harbored the least number of alleles

(DYS426, DYS448). Validation of the 20 Y-STR

multiplex system on population samples also made it

possible to make “Allelic Ladder” so that correct and

accurate alleles calls could be made when more

samples are analyzed. For this, amplified products

were pooled for making the ladder, thus facilitating

further typing of all possible alleles by using

Genotyping Macro. The allelic profile of the Y-STR

multiplex run on 3100 genetic analyzer is represented

in Fig. 4.

Conclusions The validated Y-STR multiplex assay offers

exclusively single tube reaction, which is rapid,

reliable, reproducible, highly sensitive and cost-

effective system with a high discriminatory potential.

The increased number of loci in the multiplex

improves the power of discrimination between

individuals, which is one of the most important

requirements for individual identification and

paternity testing in forensic genetics. The

simultaneous amplification of many loci in the same

reaction not only saves time and resources but also

provides a larger dataset for comparison as compared

to tedious protocols of single locus amplifications or

tetra- and pentaplexes. The existence of a large

multiplex will also allow inclusion of statistical power

to DNA evidences as rapid collection of Y-STR

haplotye data from Indian populations is

accomplished. This is because the utility of Y-

chromosome in forensic casework requires large

population genetic samples to be studied.

Approximately 1300 male samples belonging to

different regions of the country with varied ethnicity

and linguistic affinity have already been examined

with this multiplex system. This will not only be of

assistance to justice delivery system but also provide

time estimates of various demographic events that

have been experienced by the Indian populations,

Fig. 4—Allelic ladder constructed from the multiplex PCR amplification of 20 Y-STRs in approx 900 male individuals.


48

thereby increasing our understanding of the Indian

scenario of male evolutionary and population

genetics.

Acknowledgement

We acknowledge the facility provided at Central

Forensic Science Laboratory, Kolkata, and would

specially like to thank Dr R Trivedi for providing

technical support required in the study, and to the

research scholars of the institute for carrying out

validation studies on population samples. This study

was funded by a research grant under the X Five Year

Plan to CFSL, Kolkata. SS was supported by a

fellowship from Ministry of Home Affairs, Govt of

India.

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validation of a single tube fluorescent multiplex assay

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