Forensic Science International: Genetics 12 (2014) 192–198

Evaluation of a novel material, Diomics X-SwabTM, for collectionof DNA

Pamela L. Marshall a,*, Monika Stoljarova a,b, Bobby L. Larue a,Jonathan L. King a, Bruce Budowle a,c

a Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center,

3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USAb Centre for Biology of Integrated Systems, Tallinn University of Technology, Akadeemia tee, Tallinn, Estoniac Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia

A R T I C L E I N F O

Article history:

Received 14 February 2014

Received in revised form 28 May 2014

Accepted 29 May 2014

Keywords:

Diomics X-SwabTM

Copan 4N6FLOQSwabsTM

DNA collection

DNA recovery

DNA quantitation

STR typing

A B S T R A C T

Success of DNA typing is related to the amount of target material recovered from an evidentiary item.

Generally, the more DNA that is recovered, the better the chance is of obtaining a typing result that will

be robust and reliable. One method of collecting stain materials is by swabbing. Recovery of DNA from a

number of commercially available swabs is not an efficient process. The X-SwabTM (Diomics Corporation,

La Jolla, CA) is a unique bio-specimen collection material with highly absorptive properties and can be

dissolved during certain extraction conditions. Therefore, more DNA may be collected from a substrate

and be released from the swab matrix than other swabs. The ability to recover DNA from X-Swab

material and success in STR typing were compared with the Copan 4N6FLOQSwabTM (Brescia, Italy), a

device which utilizes a proprietary flocked-swab technology to maximize DNA collection and elution

efficiency. Both types of swabs were impregnated with known amounts of DNA and body fluids and

allowed to air dry. In addition, blood was placed onto glass slides, allowed to dry and collected using both

types of swabs. DNA recovery was assessed by DNA quantitation and by STR typing. Results suggested

that X-Swab material yielded greater DNA recovery, particularly of low quantity samples (defined as

diluted neat samples), compared with the 4N6FLOQSwab. Results also indicated that X-Swab material

itself enhances yield of PCR products.

� 2014 Elsevier Ireland Ltd. All rights reserved.

Contents lists available at ScienceDirect

Forensic Science International: Genetics

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate / fs ig

1. Introduction

The first step in the process of forensic DNA typing is thecollection of evidentiary samples. Swabbing is one of severalapproaches for collecting biological evidence from stains. Success-ful recovery of DNA relies on two qualities of a swab, i.e.,absorption and adsorption. The two features impact the ability tocollect materials from a stain or surface and then release the cells/DNA during the extraction process. Swabs that are proficient atcollecting materials often are less efficient at releasing DNA fromthe swab matrix, and vice versa. Indeed, it is well-known thatrecovery of DNA from a swab is inefficient [1–3]. In fact, vanOorschot et al. [2] suggested that a significant proportion of DNA(20–76%) that is collected by a cotton cloth/swab is lost during theextraction phase which may be attributed to the collecting agent(swab, cloth, etc.) and the condition of the sample.

* Corresponding author. Tel.: +1 817 735 2940; fax: +1 817 735 5016.

E-mail address: pamela.marshall@unthsc.edu (P.L. Marshall).

http://dx.doi.org/10.1016/j.fsigen.2014.05.014

1872-4973/� 2014 Elsevier Ireland Ltd. All rights reserved.

Prinz et al. [4] compared several collection devices in anattempt to improve cell/DNA removal from fingerprint evidence.A total of 109 touched objects, including 30 single fingerprintsamples, were extracted and most of these samples provided lessthan 100 pg of DNA. However, the authors indicated that a‘‘specialized’’ swab yielded up to 2.5 times more allelescompared with cotton and Dacron swabs. Hansson et al. [5]studied different swabs as well as minitape to determine the bestcollection device for low copy number (LCN) DNA recovery. Theauthors tested a cotton DryswabTM (Medical Wire, Corsham,Wiltshire, England), Copan 4N6FLOQSwabTM (Brescia, Italy), aself-saturating swab (Puritan Medical Products Co., LLC, Guilford,ME), and the Scenesafe FASTTM minitape (Scenesafe Ltd.,Burnham on Crouch, Essex, England). While the results showedno difference in DNA recovery among the swabs, the minitapeyielded more DNA and gave full profiles for all six experimentalsamples. The DNA recovered from flocked swabs produced onlypartial profiles, while the DNA from the self-saturating swab andthe Dryswab produced three and four full profiles, respectively.Hansson et al. [5] suggested that investigators may want to

P.L. Marshall et al. / Forensic Science International: Genetics 12 (2014) 192–198 193

consider the type of substrate when choosing a collection deviceand also recommended further investigation into collectiondevices.

Brownlow et al. [6] studied DNA retrieval from a traditionalcotton swab compared with that of the 4N6FLOQSwab using threedifferent extraction platforms. The results suggested that whileboth swabs recovered high percentages of DNA (greater than 50%),the extraction platform chosen had an impact on DNA recovery.Thus, the authors recommended careful consideration of anextraction method with the choice of swab. A more recent studycarried out by Dadhania et al. [7] evaluated the 4N6FLOQSwab andcotton swab for DNA recovery using two different magnetic beadtechnologies (PrepFiler1 (Life Technologies) and DNA IQTM

(Promega Corporation, Madison, WI)). It was determined thatthe 4N6FLOQSwab yielded higher recovery of DNA using PrepFilercompared with cotton swabs yet the 4N6FLOQSwab was incom-patible with the DNA IQTM lysis buffer.

While the above studies suggested that the 4N6FLOQSwabyielded more DNA over the traditional cotton swab, recovery ofDNA often was not greater than 50%. The novel collection material,X-SwabTM (Diomics, La Jolla, CA), was selected for potentiallybetter DNA recovery as it has high absorptive qualities and can bedissolved under certain DNA extraction conditions (e.g., 1 hincubation at 56 8C). Thus, more DNA may be released from itsmatrix than other collection devices. Improving DNA yield fromcollection devices may result in some samples that traditionallyyield too little DNA becoming suitable for routine analysis.Furthermore, this device may improve retrieval of higher qualityDNA as longer stranded molecules may be released from thematrix than with other collection devices.

2. Materials and methods

2.1. Samples

2.1.1. Purified DNA

Some experiments were performed using the Quantifiler1

Human DNA Quantification Kit (Life Technologies) DNA Stan-dard (Raji cell line; 200 ng/mL purified DNA). The standard wasdiluted in water to final amounts of 1 ng, 2 ng, and 5 ng in a10 mL total volume. Negative controls were included with eachexperiment.

2.1.2. Whole blood and saliva

Whole blood and saliva were provided by University of NorthTexas Health Science Center (UNTHSC) faculty and staff. Allsamples were collected with informed consent and wereanonymized to ensure the privacy of the contributing subjectsin accordance with UNTHSC IRB. Dilutions of the whole blood(1:10, 1:50, and 1:100) and saliva (1:10, 1:50, and 1:100) wereprepared in 10 mM phosphate buffered saline (PBS). Negativecontrols were included with each experiment.

2.2. Swabs

2.2.1. X-Swab material

Samples of the X-Swab DiomatTM material (kindly provided byDiomics Corporation) were cut into squares (prototype format)weighing approximately 15 mg and measuring approximately5 mm � 5 mm. Negative controls (no DNA on swab material) wereperformed with every extraction.

2.2.2. 4N6FLOQSwab

The 4N6FLOQSwabswab was purchased from Life Technologies.Negative controls (no DNA on swab) were performed with everyextraction.

2.3. DNA extraction

2.3.1. Kits

DNA was extracted using either a modified QIAamp1 DNAMicro (Qiagen, Valencia, CA) extraction protocol using MinElute1

spin columns or PrepFiler ExpressTM DNA Extraction Kit (LifeTechnologies) according to manufacturer’s instructions. The DNAextracts were stored at 4 8C and �20 8C for short- and long-termstorage, respectively. X-Swab material was completely solubilizedfollowing the modified QIAamp extraction. For the PrepFilerExpress, nominal remnants of the material were visible but themajority of the material was solubilized.

2.3.2. Phenol:chloroform organic extraction

Extraction was performed using a standard protocol forphenol:chloroform organic extraction [8]. The DNA extracts werestored at 4 8C and �20 8C for short- and long-term storage,respectively. While there was some solubilization of the material,it was less than for the other extraction methods.

2.3.3. DNA quantification

The quantity of extracted DNA was determined using a reducedvolume (10 mL) protocol of the Applied Biosystems1 QuantifilerTM

Human DNA Quantification Kit (Life Technologies) on an AppliedBiosystems1 7500 Real-Time PCR System (Life Technologies) withproper controls.

2.3.4. Amplification

For the AmpF‘STR1 Identifiler1 Plus kit (Life Technologies),thermal cycling was performed on a GeneAmp1 PCR System 9700(Life Technologies) using 28 or 29 cycles (if quantity of DNA wasbelow 0.5 ng) according to the manufacturer’s recommendations.For the PowerPlex1 ESI 17 Pro System (Promega Corporation,Madison, WI), thermal cycling was performed on a GeneAmp1 PCRSystem 9700 according to manufacturer’s recommendations.Positive (9947A), negative (no template DNA), and reagent blankcontrols were included on each assay plate.

2.3.5. Capillary electrophoresis and analysis

Capillary electrophoresis was performed on an AppliedBiosystems1 3130xl Genetic Analyzer (Life Technologies) usingPOP-4TM polymer (Life Technologies) following manufacturer’sinstructions. Data were analyzed using Applied Biosystems1

GeneMapper1 ID v3.2 software (Life Technologies).

2.3.6. RapidHITTM human DNA identification system (IntegenX,

Pleasanton, CA)

10 mL of whole blood from five individuals were applied to X-Swab material and allowed to dry overnight. The samples were runon the RapidHIT system according to the manufacturer’s instruc-tions. DNA results were analyzed with GeneMarker HID v2.4.0(SoftGenetics; State College, PA, USA).

2.3.7. Massively Parallel Sequencing (MPS)

A 1:10 dilution of whole blood was placed on X-Swab material,dried overnight, and extracted using the PrepFiler ExpressTM DNAExtraction Kit. The whole mitochondrial genomes from tworeplicates were sequenced using the MiSeqTM workflow (Illumina,San Diego, CA) as described by King et al. [9].

2.3.8. Data analysis

The quantity of DNA recovered from X-Swab material wasevaluated and compared with the 4N6FLOQSwab. PCR productyield (based on relative fluorescence units (RFUs)) and peak heightratio (PHR) for heterozygous loci were evaluated and comparedwith controls using a freely available in-house excel workbook,

Table 1Extraction efficiency of the X-Swab impregnated with 5 mL whole blood (N = 10)

using three different extraction methods.

Extraction method Average DNA

concentration

(ng/mL)

Standard

deviation

(ng/mL)

Elution

volume

(mL)

Total DNA

yield

(in ng)

MinElute 2.843 0.67 30 85.29

PrepFiler Express 2.676 0.63 50 133.8

Organic 0.7438 0.46 50 37.19

P.L. Marshall et al. / Forensic Science International: Genetics 12 (2014) 192–198194

PHASTR (http://web.unthsc.edu/info/200210/molecular_and_me-dical_genetics/887/research_and_development_laboratory/3).Intra-locus PHRs were calculated for a given locus by dividing thepeak height of an allele with a lower RFU value by the peak heightof an allele with a higher RFU value, and then multiplying thisvalue by 100 to express the PHR as a percentage. Unpaired 2-tailedStudent’s T-tests were used to determine whether DNA yield wasdifferent between the swabs at a significance level of 0.05.

3. Results and discussion

3.1. Evaluation of Diomics X-Swab and extraction methods

Initial experiments examined X-Swab Diomat material and theextraction method that would yield the greatest quantity of DNA.Five mL of whole blood were placed onto X-Swab material anddried overnight. The samples, in replicates of ten, were thenextracted using a modified MinElute extraction protocol, PrepFilerExpress DNA Extraction Kit, and standard organic extraction. Thequantity and quality of DNA recovered from the X-Swab materialwere evaluated and compared with controls with no swab material(i.e., equivalent amount of liquid blood). The amount of DNArecovered for each extraction method is shown in Table 1 andindicated that the PrepFiler Express yielded the highest recovery ofDNA. Samples extracted using all three extraction methodsproduced the expected STR profiles using Identifiler Plus (28cycles) with similar RFU values and peak height balance (data notshown). Due to these results, the PrepFiler Express was selected forthe rest of the study.

3.2. Comparison of X-Swab material and the 4N6FLOQSwab

The effectiveness of DNA recovery using X-Swab materialinitially was compared with that of the 4N6FLOQSwab by placingknown quantities of purified DNA from Raji cells (1 ng, 2 ng, and5 ng) in a constant volume of 10 mL on both swabs (in replicates offive) and allowing them to dry overnight. The results suggestedcomparable yields for X-Swab material and 4N6FLOQSwab with 1(0.65 ng and 0.85 ng, respectively, p value = 0.54) and 2 ng of inputDNA (0.95 ng and 1.1 ng, respectively, p value = 0.78). However,with 5 ng of input DNA, X-Swab material had an average DNA yieldof (3 ng � 1) total DNA, which was twice as much DNA as recoveredwith the 4N6FLOQSwab (1.6 ng � 0.65). The difference in DNA yieldwas not significant but was approaching significance (p val-ue = 0.053). Placing purified DNA on swabs does not necessarilydemonstrate extraction efficiency as would using cellular material

Fig. 1. Average DNA yield for whole blood and saliva samples. A dilution series of two di

1:50, and 1:100) in 10 mM PBS was prepared. 10 mL of each sample were placed on bot

whole blood and panel (b) shows average DNA yield for saliva. Significant differences

(see below). However, the comparable results with purified DNAsuggests that the novel Diomat polymer does not interact with DNAmore so than does 4N6FLOQSwab material.

Known quantities of whole blood (1 mL and 5 mL) were placedonto X-Swab material and the 4N6FLOQSwab (in replicates of five)and allowed to dry overnight. While a comparable mean DNA yieldwas observed with 5 mL (115 ng � 19 and 105 ng � 51, p val-ue = 0.69) for X-Swab material and 4N6FLOQSwab, respectively, theaverage DNA yield for 1 mL with X-Swab material was (57 ng � 15)compared with (37 ng � 8) DNA recovery from the 4N6FLOQSwab.However, the difference in DNA yield for 1 mL was not significant (pvalue = 0.24).

Dilutions of whole blood (1:5, 1:10, 1:50, and 1:100) wereprepared in 10 mM PBS and10 mL placed onto the swabs andallowed to dry overnight. The 4N6FLOQSwab yielded slightly betterDNA recovery at the 1:5 dilution (p value = 0.13) (Fig. 1a). However,X-Swab material had a significantly higher DNA recovery at the 1:10(p value = 0.008) and 1:50 dilutions (p value = 2.1 � 10�7). The X-Swab material had a higher DNA recovery at the 1:100 dilution butthe difference in yield was not significant (p value = 0.55). Oneexplanation for the comparable results at the greatest dilution ofblood tested is that the amount of recoverable DNA is approachingthe limit of detection for both samples. These overall resultssuggested that X-Swab material holds promise as an effective toolfor low template DNA recovery.

Based on the greater, although not significantly, yield at the1:100 dilutions, a larger sample size (N = 100) evaluation at wholeblood 1:100 dilution was carried out solely on X-Swab material toassess range of performance at this lower level DNA amount. Theaverage percent recovery was (80% � 5) for the X-Swab materialwhen DNA yield was compared with the controls with no swabmaterial. These data were consistent with results previously obtainedin the smaller-scale study and indicated that a large proportion of theDNA may be recovered from lower quantity DNA-type samples.

Next, a series of saliva dilutions (1:5, 1:10, 1:50, and 1:100)were placed onto the swabs and allowed to dry overnight. A

fferent sample types, whole blood (1:5, 1:10, 1:50, and 1:100) and saliva (1:5, 1:10,

h 4N6FLOQSwaband X-Swab (10 replicates). Panel (a) shows average DNA yield for

are marked by * for p < 0.05 and ** for p < 0.01.

Fig. 2. Average RFU values of dilutions of whole blood. 10 mL of 1:10 and 1:100 dilutions of whole blood were placed on glass slides and allowed to dry. PBS was used to

moisten X-Swab (10 mL) and 4N6FLOQSwab (30 mL) and the stains on slides were collected (10 replicate slides for each swab type). All ten DNA extracts for the 1:10 dilution

stains from both swab types were normalized to 1 ng. For the 1:100 dilution, only seven X-Swab and four 4N6FLOQSwab samples yielded sufficient DNA to be normalized to

1 ng, and thus, only these samples were used for STR comparison. Extracts were amplified using the PowerPlex1 ESI 17 Pro System. Panel (a) shows average RFUs for 1:10

dilution and panel (b) shows average RFUs for 1:100 dilution.

P.L. Marshall et al. / Forensic Science International: Genetics 12 (2014) 192–198 195

significantly higher DNA yield was observed for X-Swab materialfor the 1:5 (p value = 1.1 � 10�7) and 1:10 dilutions (p val-ue = 0.002) compared with DNA recovery from the 4N6FLOQSwab(Fig. 1b). DNA yields from the 1:50 (p value = 0.26) and 1:100 (p

value = 0.23) saliva dilutions were comparable. The likely expla-nation of similar results at the greater dilutions of saliva is theamount of recoverable DNA is approaching or has reached a limit ofdetection and thus no conclusions on efficiency comparisons canbe gleaned at 1:50 and 1:100 dilutions.

3.3. Stain study

DNA recovery from X-Swab material and the 4N6FLOQSwabwere compared with laboratory-prepared stains. Using twodilutions of whole blood (1:10 and 1:100), 10 mL were placedon glass slides, in replicates of 10, and allowed to dry (for three

days). Because of the different size and surface area between the X-Swab material and the 4N6FLOQSwab, different volumes ofwetting agent were required. It was determined that 10 mLproperly wetted the X-Swab while 30 mL were necessary for the4N6FLOQSwab. For the 1:10 dilution stains, the X-Swab and the4N6FLOQSwab had very similar average DNA yields (62 ng � 17and 73 ng � 27, respectively; p value = 0.82), even though the X-Swabsurface area and volume were considerably smaller. For the 1:100dilution stains, the X-Swab yielded nearly twice as much DNA(6.6 ng � 1.5) as the 4N6FLOQSwab (3.6 ng � 1) but the difference inyield was not significant (p value = 0.30). There was sufficient DNArecovery for all ten DNA extracts from the 1:10 dilution stains for bothswab types to be normalized to 1 ng. For the 1:100 dilution, onlyseven X-Swab and four 4N6FLOQSwab samples were able to benormalized to 1 ng and thus, only these samples were used for STRcomparison. Partial or full STR profiles were obtained for all samples

Fig. 3. Average RFU and PHR values of a 1:10 dilution of whole blood. 10 mL of a 1:10 dilution of whole blood were placed on both the 4N6FLOQSwab and X-Swab (10

replicates). Swabs were extracted using PrepFiler Express. 1 ng of DNA was amplified with the AmpF‘STR Identifiler Plus kit (28 cycles). Panel (a) shows average RFUs and

panel (b) shows average PHRs.

P.L. Marshall et al. / Forensic Science International: Genetics 12 (2014) 192–198196

for both swab types at both dilutions using the PowerPlex ESI 17 ProSystem. For both the 1:10 and 1:100 dilutions, the X-Swab yieldedhigher RFU values at all loci (Fig. 2a and b, respectively). For the 1:10dilution, DNA extracted X-Swab material yielded an overall averagepeak height RFU (all loci) of 6096 � 304 compared with 4452 � 309for the 4N6FLOQSwab. For the 1:100 dilution, the overall averagepeak height RFUs for X-Swab material and 4N6FLOQSwab were5683 � 469 and 4163 � 579, respectively. There were no observabledifferences in the PHR of the 1:10 or 1:100 dilutions comparisons.

3.4. An examination of the X-SwabTM polymer

STR typing results suggested that DNA extracted from X-Swabmaterial tended to yield increased peak heights compared withDNA from the 4N6FLOQSwab. When DNA extracted fromboth swabs was normalized to 1 ng and then typed for STRs withIdentifiler Plus (28 cycles), X-Swab material consistently yieldedhigher RFUs at all loci, some substantially higher, when comparedwith DNA from the 4N6FLOQSwab (Fig. 3a). No differences inaverage PHR were observed between the DNA from X-Swabmaterial and the 4N6FLOQSwab (Fig. 3b).

One possible explanation for the increase in peak heights maybe that the Diomat polymer, which was co-purified with the DNA,could be associated with the signal increase (although one cannotpreclude that some other soluble component could possibly be co-purifying as well). To test whether the solubilized polymer fromthe X-Swab material may be affecting PCR yield, clean X-Swab

Fig. 4. Comparison of water and X-Swab extract on amplification of a 500 pg whole blood s

and 500 pg were placed in a PCR. Either 9 mL of water or X-Swab extract were added. Am

Panel (a) shows average RFUs and panel (b) shows average PHRs.

material (i.e., no DNA) was subjected to the DNA extractionprotocol. DNA (500 pg) from liquid whole blood and either 9 mL ofsterile water or X-Swab extract were placed in Identifiler Plusamplification reactions (29 cycles). The PCRs with X-Swab extractyielded higher RFU values at all loci compared with those withwater (Fig. 4a) and no differences were observed in the averagePHR (Fig. 4b). The same experiment was carried out except theamount of template DNA was reduced to100 pg. While the resultswere not as pronounced, samples with the X-Swab extract yieldedhigher RFU values at 13 of the 16 loci compared with only water asan additive (Fig. 5a) and no differences were observed in theaverage PHR (Fig. 5b). These results supported the hypothesis thatthe presence of the X-Swab extract in the PCR increased PCR yield.

3.5. Compatibility of the X-Swab material with new technologies

Although preliminary, X-Swab material was compatible withalternate technologies, such as the RapidHITTM Human DNAIdentification System and Massively Parallel Sequencing (MPS).Samples were run on the RapidHIT system according to themanufacturer’s instructions. Fig. 6 shows a representativeelectropherogram. All five samples produced the expected DNAprofiles, which demonstrated compatibility of a blood impregnat-ed X-Swab material with the RapidHIT system.

Because of the potential of the X-Swab polymer to co-purifywith extracted DNA in the protocols described herein, the effectsthat polymer may have with MPS technology were tested.

ample. A whole blood sample was extracted using PrepFiler Express and normalized

plification was performed using with the AmpF‘STR Identifiler Plus kit (29 cycles).

Fig. 5. Comparison of water and X-Swab extract on amplification of a100 pg whole blood sample. A whole blood sample was extracted using PrepFiler Express and normalized

and 100 pg were placed in a PCR. Either 9 mL of water or X-Swab extract were added. Amplification was performed with the AmpF‘STR Identifiler Plus kit (29 cycles). Panel (a)

shows average RFUs and panel (b) shows average PHRs.

Fig. 6. Representative electropherogram of a 10 mL whole blood sample applied to X-Swab and analyzed on RapidHIT Human DNA Identification System. DNA results were

analyzed with GeneMarker HID v2.4.0.

P.L. Marshall et al. / Forensic Science International: Genetics 12 (2014) 192–198 197

Haplotypes were generated for the two samples that had beensequenced previously [9]. The range of coverage (753-17851X) andbase calls were concordant. These results suggested that DNAextracted from X-Swab material was compatible with MPStechnology.

4. Conclusions

Most collection swabs are inefficient in yielding DNA; that is,portions of DNA remain entrapped in the swab. This residuallyretained DNA can be relatively substantial. The results in this studydemonstrated that X-Swab material yielded more DNA and higher

average peak heights compared with DNA extracted from the4N6FLOQSwab for both blood and saliva samples. With a greaterrecovery of DNA, X-Swab material offers a potential solution in thatthere may be more ‘‘low level’’ samples that could yield sufficientDNA quantity for conventional STR typing protocols. Results alsoindicated that X-Swab material itself enhances yield of PCRproducts. X-Swab material was compatible with multiple extrac-tion methodologies, although some performed better regardingyield than others. It is possible that extraction methods could bemodified to yield more DNA and that shall be part of future studies.Lastly, X-Swab material and the DNA recovered were well-suitedwith the newer technologies of RapidHIT and MPS.

P.L. Marshall et al. / Forensic Science International: Genetics 12 (2014) 192–198198

Ethical standards

The work described above was performed in accordance withall laws (both Federal and State) that apply to research, researcherconduct, and the protection of human test subjects. We alsooperate under the guidance of and in accordance with the policiesof the UNTHSC Institutional Review Board.

Conflict of interest

All authors declare that they have no conflict of interest.

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