str genotyping

8/8/2019 STR Genotyping

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86 K. Lazaruk et a/.

Catherine Lazaruk'. Sean Walsh'rank OaksIennis Gilbert3arnett B. Rosenblum

Steve Menchen)on Scheibler

3. Michael Wenz:ydne Holtleanette WallinPE Applied Biosystems,Foster City, CA, USA

Electrophoresis 1998, 19, 86-93

Genotyping of forensic short tandem repeat (STR)

systems based on sizing precision in a capillaryelectrophoresis instrument

Automated fluorescence analysis of polymerase chain reaction (PCR)-ampli-fied short tandem repeat (STR) systems by capillary electrophoresis (CE) isbecoming an established tool both in forensic casework and in the implemen-tation of both state and national convicted offender DNA databases. A newcapillary electrophoresis instrument, the ABI Prism 3 10 Genetic Analyzer,along with the Performance Optimized Polymer 4 (POP-4) provides an auto-mated and precise method for simultaneously analyzing ten fluorescentlylabeled STR loci from a single PCR amplification kit, which provides a powerof discrimination of approximately one in five billion from a single PCR ampli-fication. Data are presented on sizing precision, sizing accuracy, and resolutio

nfor the STR loci in the AmpFlSTR ProfilerTM kit. Sizing accuracy is highlydependent on the electrophoresis system, and therefore the reporting ofalleles based on the nucleotide size obtained from an electrophoresis systemis not recommended for forensic work. The precision of the 310 capillary elec-trophoresis system, coupled with software developed for automated genoty-ping of alleles based on the use of an allelic ladder, allows for accurate genoty-ping of STR loci. Sizing precision of 5 0.16 nucleotide standard deviation wasobtained with this system, thus allowing for accurate genotyping of length vari-

ants that differ in length by a single nucleotide.

1 Introduction

Automated fluorescence analysis of PCR-amplified shorttandem repeat (STR) loci is rapidly becoming a powerfultool in forensic casework and databanking applications[l, 21. The STR markers of choice are predominantlytetranucleotide repeat loci that have a polymorphicnumber of repeat units in human populations [3]. Multi-plex amplification of several STR loci in a single PCRcan produce profiles with average matching probabilitiesas low as one in several billion [4]. In automated fluores-

cence analysis the alleles from an STR locus are PCR-amplified from human genomic DNA using an unla-beled primer and one primer labeled at the 5' end with a


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fluorescent dye. Denatured PCR products are then co-analyzed by electrophoresis with an in-lane size standard(DNA fragments of known size labeled in a differentcolor dye) on slab gel or capillary instruments capable ofreal-time multicolor fluorescence detection [5]. The col-lected data are then analyzed by software which automa-tically determines allele sizes based on a standard curve

for the in-lane size standard.

Capillary electrophoresis is becoming an established andsometimes preferred method for detection of fluores-cently labeled PCR products [6-101. Recently, a newcapillary electrophoresis instrument capable of simulta-neous multicolor detection and high resolution of DNA

Correspondence: Dr. Katherine Lazaruk, PE Applied Biosystems, 850Lincoln Centre Drive, Foster City, CA 94404, USA (Tel: +650-638-5486; Fax: +650-638-6333; E-mail: [email protected])

Nonstandard abbreviations: nt, nucleotide; STR,short tandem repeat

Keywords: Capillary electrophoresis / Forensic / Short tandem repeats/ Genotyping / AmpFlSTR

fragments was developed. This instrument, the ABIPrism 310 Genetic Analyzer, is highly automated. Multi-plex STR amplification products (held in a 48- or 96-welltray) are sequentially injected into a single capillary anddetected as they move past a laser detection windownear the end of the capillary. The laser-induced fluores-cence is detected on a CCD camera, which simulta-neously detects all wavelengths from 525-680 nm. The

instrument automatically reloads fresh polymer into thecapillary between injections. Each capillary has a lifetimeof at least one hundred injections; 96 samples in a singletray can therefore be analyzed by the instrument whilecompletely unattended by the user.

The polymer that is used on the 310 instrument, Per-formance Optimized Polymer 4 (POP-4, PE Applied Bio-systems), has many performance features that are criticalto the success of STR analysis in forensic casework. ThePOP-4 polymer was developed to meet the followingspecifications: detection of alleles differing in size by asingle base (up to 250 bp in length), sizing precision(between alleles of the same length) of less than 0.15nucleotide (nt) standard deviation, analysis time persample of less than 30 min, and capillary life of at least100 injections. The POP-4 polymer is run in 50 kmuncoated capillaries; the polymer dynamically coats thecapillary walls to reduce electroendosmotic flow and pre-vent loss of resolution. The POP-4 polymer was alsodesigned to provide a highly denaturing environment forthe DNA samples [ll, 121. The polymer solution consistsof linear dimethylacrylamide, 8 M urea, 5 Oh 2-pyrrolidi-none, and 1 mM EDTA, and the run temperature is setat 60 "C. These high denaturing conditions are important

in keeping the various STR alleles uniformly denatured,and are therefore important in obtaining reproduciblesizing results from one injection to the next.


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0WILEY-VCH Verlag GmbH, 69451 Weinheim, 1998 0173-0835/98/0101-0086 $17.50+.50/0


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Results indicate that the sizes obtained for STR allelescan differ depending on the gel and electrophoresis con-ditions, and depending on the instrument platform used.For example, sizes obtained for a particular allele on the

ABI Prism 377 DNA sequencer are different by up to4 bp when run on a 36 cm, 5% Long Ranger gel vs. a12 cm, 6.5% polyacrylamide gel. Differences in allelesize are also observed for samples run on the ABI Prism377 compared to the ABI Prism 310. Results also indi-cate that allele sizes can differ depending on the in-lanesize standard that is used [13]. Finally, allele sizes ob-tained under most conditions are often not the same asthe actual nt length, as determined by sequencing of thealleles. To avoid the dependence of calculated allele sizeon all of the conditions noted above, a method of accu-rate genotyping is described which takes advantage of

the ability of the in-lane size standard to effectively nor-malize mobility differences between lanes on a gel, orbetween injections on a capillary. Sample allele sizes ob-tained within a gel (on the ABI Prism 377) or within aset of capillary injections (on the ABI Prism 310) arereproducible, with standard deviations typically less than

0.15 nt.A critical component of the described method for ac-curate genotyping is the use of an allelic ladder. Theallelic ladder contains the common alleles for a parti-cular STR locus and is labeled with the same dye as thesample alleles. The alleles in the allelic ladder have

been sequenced, and therefore the number of repeatunits is known for each allele (Lazaruk et al., in prepara-tion). On the ABI Prism 310 instrument, the allelicladder is run along with the in-lane size standard inone injection, and sample alleles with in-lane sizestandard are run in all other injections on the capillary.Sample allele sizes are then compared to allelic ladderallele sizes. Sample alleles that size within 0.5 bp ofan allelic ladder allele are then assigned the appro-priate allele designation [14, 151; the nomenclature

system for allele designation is based on the numberof repeat units contained in the allele. Thus, this meth-od of genotyping is a floating bin approach, where theallele size bins for each set of capillary injections aredefined according to the sizes obtained for the allelicladder run in the same set of injections. Genotyper 2.0software automates the genotyping process by recog-nizing the injection containing the allelic ladder, andcreating new size bins for each set of samples run on acapillary.

Although most tetranucleotide STR alleles differ in sizeby four nucleotides, some common and some rarealleles do exist that differ in size by only a single nucleo-

tide relative to other alleles ([16, 171, Lazaruk et al., inpreparation). Accurate allele assignment for these allelesdepends on adequate sizing precision and peak resolu-


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tion; both of these requirements are demonstrated withallele sizes of 100-300 bp on the ABI Prism 310. Allelesat an STR locus that are the same size can sometimesdiffer in their sequence in the repeat region [18, 191, orflanking region. Results with two alleles at the vWAlocus that are the same nucleotide length, but that differin sequence at six positions, indicates that the calculated

sizes are equivalent.

Precision in genotyping forensic STRs by CE 87

This paper reports the results of severaI experimentswhich demonstrate that the ABI Prism 310 Genetic Ana-lyzer and POP-4 polymer together provide a platform foraccurate and reliable analysis of STR alleles in forensicapplications. In addition to correct allele assignment ofcommon alleles, variant alleles that differ in length froma common allele are sized precisely; alleles that differ insequence, but that are the same length as a common

allele, are also precisely defined.

2 Materials and methods

2.1 AmplificationMany experiments were carried out during the develop-ment phase of the AmpFlSTR ProfilerrM Kit (PE AppliedBiosystems, Foster City, CA) and thus some of the pa-rameters differ from the final PCR amplification param-eters and from the final ABI PrismTM 310 (PE AppliedBiosystems) electrophoresis conditions. STR alleles wereamplified using a prerelease version of the AmpFISTRProfiler PCR Amplification Kit. In a single amplification

tube this kit amplifies the STR loci: D3S1358 [20], vWA[21], FGA [22], all labeled with 5-FAM, THO1 [23], TPOX[24], CSFlPO [25], all labeled with JOE; D5S818 [26],D7S820 [27], and D13S317 [26], all la6eled with NED;and the amelogenin gender-determining locus [28], la-beled with JOE. Each of the loci labeled with a parti-cular dye are nonoverlapping in their allele size ranges.DNA (1.5 ng) from two different sets of individuals,PC1-PC47 and PC42-PC85, in a Caucasian populationdatabase was amplified using the AmpFlSTR Profiler Kitprimer set (one of each primer pair is labeled with a fluo-rescent dye), AmpFlSTR PCR Reaction Mix, andAmpliTaq GoldTM DNA Polymerase (PE Applied Biosys-tems) in a GeneAmp PCR System 9600 (Perkin-Elmer):95"C, 11 min preincubation; followed by 28 cycles of94"C, 1 min; 59"C, 1 min, 72"C, 1 min; and a final exten-sion at 60C for 45 min.

2.2 Sample preparation and electrophoresisSample preparation for capillary electrophoresis was asfollows: 1 pL of PCR product was mixed with either1 pL of Genescan-350 [ROX] (GS-350) size standard or

0.5 pL of Genescan-400HD [ROX] (GS-400HD) sizestandard and 24 pL of deionized formamide. The sam-

ples were mixed by pipeting up and down after additionof the PCR product. The samples were then denaturedat 95C for 2-3 rnin and snap-cooled in an ice-water


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formamide. The samples were then denatured at 95Cfor 2-3 min and snap-cooled in an ice-water bath. Thesample mixture (1.5 pL) was loaded into each lane ofthe 50-lane gel and run using the GS Run 361;-2400

module (3000 V, 51C).

2.3 Data analysisData from both the 310 and 377 were analyzed usingABI Prism GeneScan 2.1 software using the localSouthern sizing and light smoothing algorithms. The250 nt peak in the GS-350 size standard was not definedin the sizing curve. The reason for this is that the 250 ntDNA fragment has shown anomolous migration undersome less stringent denaturing conditions on the CE[13], and it sizes at approximately 246 nt with the POP-4polymer. The allele size data, from both the allelic lad-

ders and unknown samples, generated by the GeneScan

2.1 software, were automatically genotyped by importingthe data into the Genotyper 2.0 DNA fragment analysissoftware. Genotyper 2.0 was also used to generate atable of size values for each allele in a sample, and thistabular data was then used for further statistical analysis.3Results

3.1 Sizing accuracySizing accuracy was determined by comparing the calcu-lated allele size from the in-lane size standard to the

actual nt length, as determined by sequencing. Table 1compares nucleotide sizes for AmpFfSTR Blue Allelicladder alleles obtained from typical runs on a 310Genetic Analyzer with the sizes obtained from a typicalrun on a 377 DNA Sequencer, using two different sizestandards. The size data were calculated by GeneScan

2.1 software using the Local Southern sizing algorithm.The data in Table 1 demonstrate three important points.First, the size data for alleles at a particular locus canvary when the DNA fragments are run on the two dif-ferent types of instrument platforms (310 vs. 377). Forexample, a D3S1358 allele 12 sizes as 114.3 bp on a 37736 cm gel and as 111.0 bp on a 310 POP-4 run, bothusing the GS-350 size standard. Second, allele sizes canvary between gels of different length and/or composition(i.e. percent gel matrix) that are run within the same in-strument platform; for example, FGA allele 18 sized as217.2 nt on a 12 cm (6.5% acrylamide) 377 gel, and as220.3 nt on a 36 cm (5% Long Ranger) 377 gel. Third, dif-ferent size standards used within the same platform cangive sizes that differ by up to 1 nt; for example, the FGAallele 30 sized as 264.9 nt using GS-350 on the 310, butsized as 266.1 nt using the GS GS-400HD size standard.The size standard chosen can either improve or worsen

the accuracy of sizing. For example the GS-400HD sizestandard improved the sizing accuracy for FGA alleleson the 310, but worsened the accuracy for vWA alleles


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11-15 on both the 310 and 377.3.2 Sizing precisionPrecision is defined as the ability to obtain reproduciblesizing of DNA fragments from injection to injection on a

Electrophoresis 1998. 19, 86-93Table 1. Sizing accuracy on various ABI Prism instrument platforms,

and within an instrument platforma)

-

Eizz 'rue length TTiziii 17736cm ?l7 36 cm 310 310allele (+A) 1.5%acryl. 5%LR 5%LR POP-4 POP4GS-350 GS-350 ;s-400HD SS4oOHD

D3S1358

12 114 114.3 114.3 114.1 111.0 111.8

13 1 I8 118.3 118.3 118.3 115.2 116.314 122 122 2 122.4 122.3 119.1 120.515 I26 126.2 126.3 126.4 123.0 124.416 130 130.3 130.5 130.5 127.3 128.617 134 134.4 134.7 134.7 131.4 132.818 138 138.5 138.9 138.8 135.6 136.819 142 142.9 143.4 142.7 139.6 140.7

-vWAI1 I57 157.5 157.4 156.2 154.4 154.112 161 161.1 161.1 160.2 158.7 158.113 165 165.1 165.0 164.3 162.8 162.214 169 169.1 169.1 168.4 167.2 166.5

15 I73 173.0 173.0 172.4 171.1 170.416 177 176.9 176.9 176.5 175.1 174.517 181 180.9 180.9 180.5 179.1 178.618 185 184.9 184.8 184.6 183.0 182.619 189 188.9 188.9 188.6 187.0 186.720 193 192.9 192.9 192.7 190.9 190.921 197 197.0 196.9 196.8 194.9 195.1

FGA

18 219 2 17.2 220.3 219.3 216.1 216.719 223 221.2 224.4 223.5 220.2 220.820 227 225.2 228.6 227.6 224.2 224.921 231 229.2 232.7 231.7 228.2 229 222 235 233.0 236.8. 235.9 232.2 233.423 239 237.0 241.0 240.0 236.3 237.624 243 240.9 245.1 244.0 240.3 241.825 247 244.9 249.2 248.1 244.4 245.926 251 248.8 253.2 252.2 248.5 249.9

26.2253 250.7 255.1 254.3 250.6 251.927 255 252.8 257.0 256.2 252.5 254.028 259 256.7 260.9 260.3 256.6 258.0

29 263 260.7 264.9 264.4 260.8 262.030 267 ___ 268.5 264.9264.6 268.9 -266.1


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a)Column 1 lists the common alleles in the AmpFlSTR Blue allelicladder. Column 2 lists the true nucleotide (nt) size of each DNAfragment (considering full addition of non-template A nucleotideto the 3' end of the PCR product), based on sequence analysis.Column 3 lists the nt size determined on a 12 cm 6.5% acrylamidegel on the 377 DNA Sequencer with GS-350 size standard. Column

4 lists the nt size determined on a 36 cm 5% Long Ranger gel onthe 377 DNA Sequencer with GS-350 size standard. Column 5 liststhe nt size determined on a 36 cm 5% Long Ranger gel on the 377DNA Sequencer with GS-400HD size standard. Column 6 lists thent size determined on a 47 cm capillary using POP4 polymer onthe 310 Genetic Analyzer with GS-350 size standard. Column 7lists the nt size determined on a 47 cm capillary using POP4polymer on the 310 Genetic Analyzer with GS-400HD sizestandard. All sizing on the various platforms used the LocalSouthern algorithm to generate the standard sizing curve.

capillary electrophoresis instrument (or from lane to

lane on a slab gel; [6]). Sizing precision was determinedin two different modes: (i) using population databasesamples where sequence variation may exist betweensome STR alleles ([17]; Lazaruk et a!., in preparation),and (ii) by running allelic ladders from ten loci in 105consecutive injections, therefore assessing the precisionof the electrophoresis conditions on PCR products withknown DNA sequences.

The sizing precision on the POP-4 polymer, using Cauca-sian database samples and three lanes or injections ofallelic ladder, was compared to the within-gel sizing pre-cision of the 377 DNA Sequencer (Table 2). The preci-

sion obtained with the 377 XL (50-lane gel) and GS-350size standard was better than, or equal to, 0.09 ntstandard deviation (SD) for all of the AmpRSTR Profilerloci. The sizing precision using the POP-4 polymer withGS-350 size standard on the 310 was better than orequal to 0.12 nt (SD) for all of the AmpFfSTR Profilerloci. The sizing precision using the POP-4 polymer withGS-400HD size standard was better than or equal to


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Ekctrophoresis 1998, 19, 86-93 Precision in genotyping forensic STRs by CE

Table 2. Sizing precision on the 310 Genetic Analyzer and 377 DNA Sequencer")Locus AUele N377 Precisionwith GS350size

standardS.D.-310 Precisionwith GS350 sizestandard310 Precisionwith GS-4WHDsize standardMean-S.D.-Mean-S.D.-LOCUSiI377 Precision

with GS-350sizestandardS.D.-MeanstandardS.D.--310 Precisionwith GS-400HDsize standardhclugcnm XY5022106.47

112.170.030.05103.37109.020.050.05103.55109 690 060 05TPOX 67/133218.41222.490 020.02214.98218.900.020.05

215.90219.840.05


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0.078 41 226.60 0.04 222 93 0.05 223.80 006D3S1358 I2 4 114.40 0 07 111.17 0.12 111.99 0.14 9 I1 230.69 0.03 226 88 0.04 227.87 0.0413 4 118.33 0.02 11526 0.03 116.30 0.12 10 7 234.76 0.03 230.86 0.04 231.97 0.0814 2414 122.34 0.05 11925 0.05 120.56 0.08 II 20 238.85 0.04 234.82 004 236.17 0

.0615 126.41 0.04 123.27 0.08 124.64 0.06 12 6 242 93 0.04 238 83 0.03 240.52 0 0816172317130.54134.680.050.06127.37131 49

0.100.11128.75132.830 100 1313 Ii3 247.01 0.01 242.83 0.06 245.40 0.1218 16 138.87 0.05 135 67 0.05 136.99 0.06 CSFlPO 6 1 3 281 48 0.04 279.73 0.04 280.93 00619 3 143.32 0 03 139 73 0.00 141.00 0.13 7 1 3 285.37 0 02 283.79 0.03 284.82 0.08

289 22 0.01 287.82 0 02 288 81 0 09vWA 11 3 157.30 OM 154.51 0.05 154.13 0 09 293 09 0.02 291 87 0.04 292.70 0.0812 3 161.11 0.06 158.82 0.08 158.18 0 08 296.97 0.04 295.93 0.08 296.78 0 0813 3 165 07 0.05 16301 0.02 162.27 0.10 300.84 0.04 300.07 0.07 300 89 0.0514 7 169.07 0.05 167.26 0.05 166.56 0.10 304.79 0.04 304.53 0.08 304.87 0.0515 8 172.98 0.07 171.09 0.11 170.48 0.07 13 308 77 0.04 308.91 0.08 308.86 0.0616 27 176.93 0.04 175.15 0.05 174.60 0.07 14 312.74 0.04 313.31 0.08 312.86 0.0517 2425 180.91 0.05 179 13 0.07 178.70 0.08 31677 0.03 317.66 0.11 316.90 0.1018 184.91 0.04 183 07 0.08 182.82 0.0519 10 188.91 0.04 186.98 0.06 186.93 0.07 D5S818 134.60 0.06 131.14 0.04 132.530.0220 6 192.94 0.04 190.96 0.06 191.09 0.09 138.72 0.06 135.29 0 05 136.60 0.0121 3 197.01 0.02 194.86 0.02 195.33 0.07 143.25 0.06 139.42 0.06 140.61 0.11147.64 0.05 143.92 0.05 144.61 0.08FGA I81842210220.16224.310.020.0321620220.24

0.050.06217.10


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221.100.080 051112I1 3229

151.90155.830.040.04148.38152.810.050.06148.57152.590.060 05

2021 20228.47232.580.030 04224.26228.280.060 05225.22229.350.06

0.081314 !195159.66163.590.040 04157.15161.440.080.06I56 63160.720.060.0922231114236.73240.840.050.04

232.31236 360.06


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0.05233.63237.890.080 0915I

3 167 48 0.06 165.44 0.03 164.77 0.1124 14 244.99 0 03 240.44 0.07 242.55 0.07 D13S317 207.27 0.01 205 I2 0.07 206.130.0625 16 249.08 0.04 244.50 0.08 247.39 0.09 211.41 0.04 209 08 0.03 210.09 0 0626 4 253.04 0.04 248.~3 0 02 252.13 0 13 10 8 215.51 0.05 213.05 0.07 214.04 0 0626.2 3 255.06 0.04 250.59 0.01 254.47 0.09 219.69 0.06 217.08 0.07 218.05 0.09272829304

333257.01260.88264.85268.820.090.060.060.05252 63256.64

260.80264 900.050.040.020.06256 8026 1.07264.73268.340.130.160.130 121314i1! Y7223.76227.85231 970.050.05

0.05221.10225 05


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229 040 070.070.05222.00226.08230 16

0.080 050.09078820 7 i 3 NIA NIA 259.07 0.04 263.16 0.10THO1 5 3 169.65 0.06 166.98 0.05 166.26 0.04 266.46 0.03 263.13 0.07 266 70 0096 27 173 61 0.04 170.98 0.08 170.35 0.07 17 270 34 0.03 267.14 0.06 270.20 0.087 17 177.61 0.04 175 03 0.05 174.50 0 07 274 21 0.04 271.22 0.05 273.63 0.068 13 181.59 0.04 179.03 0.04 178.60 0.06 278.09 0.04 275.28 0.07 277.03 0 069 14 185.62 0.05 183.00 0 05 182.70 0 06 281.94 0.05 279.35 0.07 280.49 0 079.310-244-188.67

189.65-0.030.04-18601186.93-0.070.06-185.88186.88 -0.050.02-285.79289 67 -0.040.m-283.41287.45 -0.040.02 -284 37288.42-0.110.02 -

a) 47 AmpNSTR Profiler Caucasian database samples and 3 allelic ladders were runon each instrument. Therefore, alleles for which there areonly 3 data points were alleles contained only in the allelic ladder for that locus. The D7S820 allele 7 was not present in the allelic ladder atthe time the 377 XL gel was run and was added to the ladder for the final kit (other alleles were also added to the final AmpFISTR Yellowallelic ladder). Two different size standards, the GS-350 [ROX] and GS-400HD [ROX] (pre-release version of a higher density size standard)were compared on the 310.

0.16 nt (SD) for all of the AmpFlSTR Profiler loci. The Table 3. Sizing precision on the 310 Genetic Analyzera)0.16 nt SD occurred because one sample injection Size range (nt) BS sized Rangeof standard Range of standardLocus on 310 with WP-4 deviations(nt) with GS-deviations (nt) with

showed a loss of resolution (ie. peak broadening) at the polymer 350 size standard GS-400I-D size standardlarger allele sizes and in the larger size standard peaks.

Amelogenin 103 and 109 0.05 0.05

The data showed precision of 5 0.13 nt SD when this D3S1358 I 11-140 0.05-0.06 0.05-0.06sample was not included in the precision calculations (it 154-195 0.06-0.07 0.05-0.06

is recommended that a sample showing anomalously 2 16-265 0.06-0.08 0.06-0.07

THO1 166-187 0.06 0.04-0.05


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poor resolution be rerun by the user). All sample injec-

TPOX 214-243 0.05-0.07 0.05-0.06

tions on either side of the poor resolution injection CSFIPO 279-318 0.07-0.12 0.05-0 07

showed normal resolution. In each of the Caucasian

D5S818 131-170 0.05-0.07 0.04-0.06D13S317 205-233 0.06-0.07 0.06-0.09

database sample precision experiments, the sizing preci-

D7S820 255-293 0.07-0.10 0.06-0.08

sion of 5 0.16 nt allows a _+ 0.5 nt window to be set thatwill therefore contain greater than 99.7% (3 SDs) of all a) 105 injections of AmpFISTR Profiler allelic ladders were run in two

separate sets of capillary runs. Two different size standards, the

alleles that are the corresponding nucleotide size. The

GS-350-ROX (the ladder recommended in the ArnpFlSTR User's

overall sizing precision was similar with the two dif-

Manuals) and GS-400HD-ROX (a higher density size standard)

ferent size standards; there was no advantage observed

were compared.

to using the size standard with a greater density of frag-ments.

The dashed lines in Fig. 1 represent a k0.5 nt size binAn alternative method for the display of precision data is around the size obtained for the reference allelic laddershown in Fig. 1. The x-axis represents the base pair sizes injection. The data points shown in the figure indicate(and therefore discrete alleles) obtained from a single that all observed samplealleles size within _+ 0.5 nt ofinjection of allelic ladder, and the y-axis represents the the allelic ladder sizes.deviation of each sample allele size from the correspond-ing allelic ladder size. The sample alleles are AmpFZSTR Precision data for 105injections of AmpFlSTR ProfilerTMProfiler amplifications from 31 Caucasian database sam- allelic ladders using both the GS-350 and GSples, as well as two additional injections of allelic ladders. GS-400HD size standards showed standard deviations


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90 K. Lazaruk et a/. Electrophoresis 1998, 19, 86-93

ranging from 0.04 to 0.12 nt (Table 3). Therefore, run-to-run precision in sizing alleles of the same sequence isclose to the precision in sizing of alleles that may con-tain some sequence variation.

Genotyper 2.0 software automatically uses the nt size ob-tained for an injection of allelic ladder as the center ofthe size bin (allelic ladder allele size 5z 0.5 nt) for each

Figure 1. Graphical display of sizing pre-cision data. The y-axis shows the devia-tion (in bp) in sizing of each respectiveallele of 31 Caucasian database samplesand 2 injections of allelic ladder, from athird injection of allelic ladder. Thex-axis represents each discrete allele in

the allelic ladders (blue diamonds rep-resent loci labeled with 5-FAM, greensquares represent loci labeled with JOE,black triangles represent loci labeledwith NED). The dashed lines representthe one base window around the refer-ence allelic ladder injection. Data wassized with the GS-350 internal lane sizestandard.

Figure 2. Genotyper 2.0 results, showinga CSFlPO allele 10.3 identified as an off-ladder (OL) allele. Shown in upper panel

is a close-up view of the CSFlPO allelesin the AmpNSTR Green I allelic ladder.The dark grey bars are the * 0.5 nucleo-tide windows placed around each of theallelic ladder allele sizes. The bottompanel shows the CSFlPO 10.3 allele fal-ling outside of the window for theCSFlPO 11 allele, and therefore beingflagged as an off-ladder allele.

sample allele within a set of injections on a single capil-lary. The size bins are floating in the sense that size binsare redetermined based on the allelic ladder included ineach new set of capillary injections. One set of capillaryinjections can be defined as one tray of samples on the310 autosampler. The sample alleles will either fallwithin one of these size bins, or fall outside the bin. If asample allele falls outside the floating bin set by the


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EIecrrophoie.sis 1998, 19, 86-93

Precision in genotyping forensic STRs by CE

Figure 3. Resolution of alleles that differin size by only 1 nt. (A) Resolution of

THO1 9.3 and 10 alleles. The top paneldemonstrates the resolution of thealleles (185 and 186 nt, respectively) withno smoothing (i.e., the raw data). Thebottom panel shows the resolution afterthe light smoothing option was appliedduring analysis, using the LocalSouthern sizing method. (B) Resolutionof CSFlPO 10.3 and I1 alleles (299 and300 nt). Sample C168 is a CSFlPO 10.3,13; and sample C173 is a CSFlPO 10, 11.Each of the samples was amplified sepa-

rately, and they were mixed in the 310sample injection tube. The top paneldemonstrates the resolution obtainedwith the GS STR POP4 A (1 mL) runmodule (15 kV, 24 min). The bottompanel shows the resolution obtainedwhen the run module was modified to13 kV and run for 26 min. Each of theelectropherograms is analyzed data (lightsmoothing, local Southern sizing with

GS-350 size standard).

allelic ladder, the allele is flagged as a possible off-ladder also able to flagthe CSFlPO 10.3 allele in a mixture ofallele. Samples that size outside an allele size bin should 2 DNA samples containing CSFlPO 10, 10.3, 11, and 13be rerun at least two times to distinguish a true off-alleles, because the 10.3and 11 alleles were resolvedladder allele from a sizing outlier. from one another (Fig. 3).

Figure 2 demonstrates the ability of the Genotyper 2.0 The highly denaturing environment of the POP-4software to distinguish alleles that differ in size by only polymer allows precise sizing of DNA fragments that are1 nt. A sample containing a CSFlPO 10.3 allele (lower of the same length, but differ in sequence. To demon-panel) is flagged as a possible off-ladder allele because it strate this, vWA 14and 14 alleles (that differ in 6 ntfalls outside the floating bin set around the CSFlPO 11 positions; [18, 191) were injected 5 times each; the meanallele in the allelic ladder (upper panel). The CSFlPO nt sizes were 167.04 (rt0.07) and 167.20 (+0.02), respec-

10.3 allele was confirmed by sequencing to contain a par- tively, in this experiment. Since these sizes fall withintial repeat unit, and thus to differ in size from an 11 0.5 bp of each other, the alleles will both be labeled as

allele by one nucleotide. The Genotyper 2.0 software was vWA 14 alleles by the Genotyper 2.0 software.


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3.3 ResolutionAs noted above, an important feature of the 310 with

POP-4 polymer is the ability to resolve peaks that differin size by only 1 nt. Resolution is defined here as theability of the GeneScan 2.1 Analysis software to assignsizes to two peaks. Figure 3A shows the resolution ofthe THO1 9.3/10 alleles (sizing at approximately 186 and187 nt), with no smoothing and with the light smoothingfunction applied in the analysis. When no smoothing isapplied (which reflects the resolution of the raw datapeaks) the peaks are resolved approximately 43 O/o downto baseline (Fig. 3A, upper panel). With light smoothingapplied during the analysis, the resolution of the peaksbecomes slightly less apparent, and the peaks appear to

be resolved to baseline to a lesser degree (approximately30%, Fig. 3A, lower panel).

A mixture of CSFlPO samples containing both a 10.3and 11 allele (sizing at 299 and 300 nt) is shown in Fig.3B. The CSFlPO 10.3 and 11 alleles are resolved underthe standard running conditions of 15 kV for 24 min(Fig. 3B, upper panel). Slightly better resolution was ob-tained when the run voltage was decreased to 13 kV witha concurrent increase in the electrophoresis time to26 min (Fig. 3B, lower panel). Even under the standardconditions, the analysis software detects the inflectionpoint and calls two different peaks which can flag the

analyst to rerun the samples under conditions that allowfor better resolution of alleles at this larger size range, ifdesired.

4Discussion

Sizing precision is much more important than sizingaccuracy when comparing samples from different sour-ces, for example evidence vs. reference samples, to deter-mine whether the samples are consistent with having ori-ginated from the same source. Accuracy is defined hereas how close the calculated size is to the actual length innucleotides, as determined by sequencing. Precision isdefined here as reproducibility in sizing an allele mul-tiple times over many injections. Genotyping with theAmpFlSTR Profiler (and other AmpFISTR) STR TypingKits together with the ABI Prism instruments relies onthis precision in sizing.

At first, it might seem surprising that the same allele cansize differently on different platforms when an internallane size standard (ex. GS-350) is used in every lane ofthe gel or injection on the capillary. The observation,however, that DNA migrates differentially under dif-

ferent electrophoresis conditions, is demonstrated inTable 1. The matrix (polyacrylamide vs. Long Ranger vs.entangled polymer), buffer, urea concentration, electric


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field, temperature, etc., can all affect the relative size ofan allele as compared to a defined set of internal lanesize standard DNA fragments. Additionally, the standardcurve produced, and therefore the calculated allele size,can vary between different in-lane size standards. Allelesfrom a single individual, then, could be reported as verydifferent sizes from laboratory to laboratory, depending

on the detection system used in each laboratory. For

these reasons, the in-lane size standard is best used onlyto normalize any migration differences between injec-tions on a capillary, or between lanes of a gel. Normaliza-tion of migration results in sizing precision, as shown forthe STR alleles described in this paper.

In the AmpFlSTR PCR amplification kits, an allelicladder containing all of the common alleles at each par-ticular locus is provided. The allelic ladder is run withevery set of samples, and the resulting allelic ladder

allele sizes are used to set floating sizebins

. Since theprecision in sizing on the 310 with POP-4 polymer is

< 0.15 nt standard deviation, 99.7% of all alleles that arethe same length in nucleotides should fall within -I-0.45nt of the allelic ladder allele size [14]. The occurrence ofsample alleles falling outside the allelic ladder size bindue to measurement error will be rare, and this errorshould not be reproducible when the sample is rerun.True length variants that differ in length by a singlenucleotide will consistently fall outside the bin setaround the allelic ladder allele when the sample is rerunfor confirmation. The floating size bins are used to

assign an allele designation to all of the unknown alleles.This floating bin sizing method using allelic ladders canbe performed automatically by the Genotyper 2.0 soft-ware. It has been shown that sequence differences be-tween alleles (for example vWA 14 and 14) do not affectmobility, and therefore sizing, on the ABI PRISM 377and 310 instruments. The denaturing capacity of thesesystems ensures that the STR loci in the AmpFlSTRSTR Typing Kits are typed according to the length differ-ences between alleles. Results also demonstrate thatpeak resolution is sufficient to distinguish between STRalleles that differ in size by only a single nucleotide.In conclusion, analysis of STR alleles on the ABI Prism310 Genetic Analyzer using POP-4 polymer enables dis-crete allele assignment of all STR alleles. Objective,accurate genotyping is thus possible, and automation ofthis process using Genotyper 2.0 software will enableease of use and high throughput capabilities.

Received April 23, 1997

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