Prototype PowerPlex® Y23 System: A concordance study

Forensic Science International: Genetics 7 (2013) 204–208

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Prototype PowerPlex1 Y23 System: A concordance study Carey Davis a,*, Jianye Ge a, Cynthia Sprecher b, Abirami Chidambaram c, Jonelle Thompson b, Margaret Ewing b, Patricia Fulmer b, Dawn Rabbach b, Douglas Storts b, Bruce Budowle a a

University of North Texas Health Science Center, Institute of Applied Genetics, Department of Forensic and Investigative Genetics, 3500 Camp Bowie Blvd, Fort Worth, TX 76107, United States b Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, United States c State of Alaska, Department of Public Safety, Scientific Crime Detection Laboratory, 5500 E Tudor Road, Anchorage, AK 99507, United States

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 March 2012 Received in revised form 1 June 2012 Accepted 2 June 2012

The Prototype PowerPlex1 Y23 System (Promega Corporation, Madison, WI) is a polymerase chain reaction-based amplification kit that targets the 23 Y STR loci DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and Y-GATA-H4. A total of 951 samples from six populations were typed to evaluate the kit and examine concordance for 17 of the loci that are in common with those that can be typed using the AmpFlSTR1 YfilerTM kit (Life Technologies, Carlsbad, CA). A total of 16,167 loci were analyzed for each multiplex, and overall concordance was observed. Because of different kit designs, and although concordant for the genetic type, discordant calls can occur due to a deletion at the DYS448 locus. Users should take into consideration such nomenclature anomalies when comparing Y STR profiles. This new kit allows a large battery of Y STR loci to be analyzed using the same basic technologies already employed in forensic laboratories. ß 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Forensic DNA analysis Y chromosome STR concordance US populations Native Alaskans

1. Introduction The degree of concordance of STR allele calls among commercial kits is an important consideration for sharing and comparing STR profiles among laboratories and through national database searches. Because different primer sets for the same loci are used by the various manufacturers, discordance may occur due to primer binding site variants, deletions at primer binding sites, insertions and/or deletions in flanking regions, and sequence variation affecting secondary conformation [1–6]. Indeed, some discordant findings, such as for the D8S1179 [1] and SE33 [2–5] loci, have resulted in additional and/or new primers added to multiplexes. Therefore as new kits become available, it is desirable to assess the degree of potential discrepancies between allele calls from different constructs and whether they are sufficiently severe to warrant additional primer design. While there is no current standard for the allowable percent of discordance, this study sought to determine if there was discordance at any locus exceeding 1%. The best way to find discrepancies under the current state-of-the-art analytical conditions is to perform concordance studies on population samples.

* Corresponding author. Tel.: +1 817 735 2940; fax: +1 817 735 5016. E-mail address: [email protected] (C. Davis). 1872-4973/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2012.06.005

The Prototype PowerPlex1 Y231 System (Promega Corporation, Madison, WI) is a polymerase chain reaction-based amplification kit that targets the 23 Y STR loci DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and Y-GATA-H4 (Fig. 1). Thus, a large battery of loci can be typed simultaneously. The Prototype PowerPlex1 Y23 System has the same primer sequences and configuration as the official commercial kit, so no concordance issues should occur between the two versions of the kit. This study examines concordance among six population samples using the Prototype PowerPlex1 Y23 System and the AmpFlSTR1 YfilerTM kits. Three major US populations and three Native Alaskan populations were chosen to assess whether concordance issues are prevalent in cosmopolitan and less diverse populations in the US.

2. Methods and materials A total of 951 samples from unrelated males among six separate sample populations were analyzed for this study. The six populations were three Native Alaskan populations (Inupiat (N = 148), Yupik (N = 141), and Athabaskan (N = 152)) and three major United States populations (African American (N = 168),

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Fig. 1. Representative electropherogram displaying the Y23 haplotype profile of the NIST standard 2395 A.

Caucasian (N = 175), and Hispanic (N = 167)). The sources and types of the samples have been described previously [7,8]. The DNA was extracted using Qiagen’s EZ1 DNA Investigator Kit on the EZ1 Advanced XL (Qiagen Inc., Valencia, CA) following the manufacturer’s protocol. Samples were eluted into 40 mL of 0.1 M Tris, 0.1 mM EDTA (TE 4), and all extracted samples were stored at 20 8C until analyzed. The quantity of recovered DNA was determined using the Quantifiler1 Human DNA Quantification Kit (Life Technologies, Foster City, CA) and the ABI 7500 Real-Time PCR System (Life Technologies) according to the manufacturer’s instructions. Samples were normalized to 1.0 ng/mL with ddH2O. The DNA samples were amplified with reagents contained in the Prototype PowerPlex1 Y23 System kit (Promega Corporation, Madison, WI) according to the manufacturer’s recommendations in an ABI PRISM1GeneAmp1 9700 Silver block Thermal Cycler (Life Technologies) using the 9600 emulation mode for 30 cycles. PCR products were separated and detected on an ABI PRISM1 3130 xl Genetic Analyzer (Life Technologies) following the manufacturer’s recommendations. Samples were injected for 5 s at 3 kV and separated electrophoretically in performance optimized polymer (POP-4TM; Life Technologies) using the HIDFragmentAnalysis36_POP4 Module (Life Technologies) and a 1500 s run time. The DNA samples were also amplified with reagents contained in the AmpFlSTR1 YfilerTM kit (Life Technologies) in a reduced reaction volume of 15 mL (14 mL of master mix and 1 mL of DNA). The master mix consisted of the following components for each sample: 5.8 mL of AmpFlSTR1 PCR Reaction Mix, 2.9 mL of AmpFlSTR1YfilerTM primers, 5.0 mL of ddH2O, and 0.30 mL of 5 U/mL of AmpliTaq1 Gold DNA polymerase (Life Technologies). Amplification was performed in an ABI PRISM1GeneAmp1 9700 Silver block Thermal Cycler (Life Technologies) using the 9600 emulation mode for 30 cycles. Prior to electrophoresis, 1 mL of the amplified product or allelic ladder and 0.5 mL of GeneScanTM-500 LIZ1 size standard (Life Technologies) were added to 9 mL of deionized Hi-DiTM formamide (Life Technologies), denatured at 95 8C for 5 min, and placed on ice for 5 min. PCR products were separated and detected as described above except that a 10-s injection was used. The data were collected using the ABI PRISM1 3130 xl Genetic Analyzer Data Collection Software 3.0. Electrophoresis results were analyzed with GeneMapper1 ID software

v3.2 (Life Technologies). Allele peaks were called when the peak heights were equal to or greater than 50 relative fluorescence units.

3. Results Between the two kits there are 17 Y STR loci in common. All typing results for these Y STR loci were concordant. The Prototype PowerPlex1 Y23 System is the only currently available commercial kit for the six Y STR loci DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643; thus these loci were not evaluated for concordance in this study. However, successful typing was obtained for the six Y STRs for all 951 samples (four null alleles were observed at theDYS643 locus (see below)). While not a typing concordance difference, the design of the two commercial kits can lead to a potential nomenclature issue due to variant alleles at the DYS448 locus. A total of 13 null alleles at DYS448 were detected and concordant in both multiplex kits (12 in the Native Alaskan populations and one in the Hispanic sample population). Budowle et al. [9] previously described that a small subset of samples at the DYS448 locus carry a deletion within the amplified region that causes the resulting smaller-size amplicon to reside at a position in a smaller locus. This same scenario was observed in one Hispanic sample in this data set. For AmpFlSTR1 YfilerTM, the deletion-bearing DYS448 allele is observed in the electropherogram region occupied by the DYS437 locus (Fig. 2A). For the PowerPlex1 Y23 System, the same allele migrates with the DYS576 locus alleles (Fig. 2B). This phenomenon may result in two identical profiles being named differently. For the AmpFlSTR1 YfilerTM kit, the profiles will be labeled with two alleles at the DYS437 locus and no allele at the DYS448 locus and for the Prototype PowerPlex1 Y23 System the same profile will be labeled with two alleles at the DYS576 locus and no allele at the DYS448 locus. Such shifts were not seen for other loci in this study. However, the phenomenon may arise at low occurrences at other loci and practitioners should be cognizant of the possible nomenclature discordance. Database searching software should be modified or developed, if Y STRs are used for profile searching, to take the necessary precautions to reduce false exclusions when comparing profiles generated by these two kits.

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Fig. 2. Apparent DYS448 null allele. (A) Apparent DYS448 null allele observed in DYS437 locus for AmpFlSTR1 YfilerTM kit and (B) Apparent DYS448 null allele observed in DYS576 locus for Prototype PowerPlex1 Y23 System.

A benefit of the Prototype PowerPlex1 Y23 System is the additional loci that will increase the discrimination power of Y STR typing and because of the relatively high mutation rate of STRs increase the chance of differentiating paternal lineage relatives [10–13]. In addition, the allelic ladder ranges have been increased for all of the loci compared with the AmpFlSTR1 YfilerTM kit. Thus, fewer extrapolated allele calls beyond the allelic ladder range will occur with the PowerPlex1 Y23 System. Of the 951 samples analyzed, an allele outside of the bin set designated by AmpFlSTR1 YfilerTM had to be manually called a total of 20 times (Table 1). All of these alleles were contained within the bin sets of the Prototype PowerPlex1 Y23 System. While no alleles were observed to fall outside of the bin set provided by Prototype PowerPlex1 Y23 System, as more samples are typed it is likely that an allele(s) will be found that reside beyond the range of the allelic ladder. Regardless, with the extended range fewer alleles will be compiled within the ‘‘>’’ and ‘‘<’’ allele categories. Again when searching databases, the software should accommodate alleles that reside above and below allelic ladder ranges but are named differently (e.g., assume that the largest allele in the allelic ladder in the two kits is different – one is a 15 and the other is a 17, then >15 and >17 for a sample named with different kits should not be considered a mismatch).

For the six new Y STR loci contained within Prototype PowerPlex1 Y23 System, several new variants were observed that are not recorded in STRbase [14]. One Hispanic sample contained a duplication in both the DYS570 and DYS576 loci, yielding a 17, 18 type for both loci. Since the DYS570 and DYS576 loci reside close together on the p arm (6.7 and 7 Mb, respectively), with no other of the PowerPlex1 Y23 System loci residing between them, a likely explanation for the types at these loci is that the entire area has been duplicated [15]. Another duplication was observed at the DYS643 locus in one individual from the Hispanic population yielding a 9,10 type. There were four apparent null alleles (which have yet to be sequenced) observed at the DYS643 locus; three in African Americans and one in Hispanics. 4. Population data The six new loci found in the Prototype PowerPlex1 Y23 System were examined for diversity and discrimination power. The numbers of distinct haplotypes within and among the populations are shown in Table 2. The number of times a haplotype was observed in each population and across all six populations is shown in Table 3. The six loci haplotypes were found to be highly discriminating among each population (Table 4). Consistent with

C. Davis et al. / Forensic Science International: Genetics 7 (2013) 204–208 Table 1 Samples in AmpFlSTR1 YfilerTM that are outside the bin set but within the bin set of the Prototype PowerPlex1 Y23 System. Sample

Locus

Yfiler Call

Y23 call

Percent of allele calls changed manually per locus

CAU.10585 AFA.12150 CAU.1713 A.768.P B.2165.S B.2175.S B.2305.S B.291.S B.3171.S B.3276.S B.17684.S B.52.P B.5471.S AFA.10309 AFA.10448 CAU.1155 A.464.P B.17898.S AFA.10514 CAU.10575

DYS448 DYS456 DYS456 DYS458 DYS458 DYS458 DYS458 DYS458 DYS458 DYS458 DYS458 DYS458 DYS458 DYS635 DYS635 DYS635 Y-GATA-H4 Y-GATA-H4 Y-GATA-H4 Y-GATA-H4

<17 <13 >18 <14 <14 <14 <14 <14 <14 <14 <14 <14 >20 <19 <19 >26 >13 >13 >13 >13

15 12 19 13 13 13 13 13 13 13 13 13 21 18 17 27 14 15 14 14

0.10% 0.21% 1.05%

Table 4 Maximum six loci haplotype PD for each population and all populations combined. Population

PD

Total AFA CAU HIS Inupiat Yupik Athabaskan

0.9968 0.9928 0.9917 0.9924 0.9751 0.9759 0.9871

AFA, African American; CAU, Caucasian; HIS, Hispanic.

AmpFlSTR1 YfilerTM kit [7]. The global Fst value for the six new loci was 0.0063 for all six populations and 0.0085 for the three Native Alaskan populations combined. 0.32%

5. Data submission 0.42%

Table 2 Number of distinct haplotypes for the six new loci for each population examined. Distinct haplotypes are the number of different haplotypes within a population. Population specific haplotypes are the distinct haplotypes that were observed in only one population group. Population

Sample size

No. of distinct haplotypes

No. of population specific haplotypes

AFA CAU HIS Inupiat Yupik Athabaskan

168 175 167 148 141 152

154 152 148 76 80 109

135 113 112 48 48 73

AFA, African American; CAU, Caucasian; HIS, Hispanic.

other Y STRs [7] the major populations demonstrated a higher haplotype diversity (exceeding 0.99) compared with the less diverse Native Alaskan populations. Combining the three Native Alaskan populations, the haplotype power of discrimination (PD) reached 0.9908 with only the six new loci which compared favorably to the PD of 0.9959 observed for all 17 loci in the Table 3 Number of times a six loci haplotype was observed in each population. The combined column indicates number of times a haplotype was observed across all populations. # of counts

Combined

AFA

CAU

HIS

Inupiat

Yupik

Athabaskan

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

469 78 29 10 6 4 6 1 4 2 0 2 0 0 1

143 8 3 0 0 0 0 0 0 0 0 0 0 0 0

138 10 1 2 0 1 0 0 0 0 0 0 0 0 0

133 11 4 0 0 0 0 0 0 0 0 0 0 0 0

48 12 6 4 3 0 1 1 0 0 0 1 0 0 0

58 9 5 1 3 1 1 1 0 1 0 0 0 0 0

82 19 4 2 0 2 0 0 0 0 0 0 0 0 0

AFA, African American; CAU, Caucasian; HIS, Hispanic.

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This paper follows the recommendations of the ISFG on the use of Y-STRs in forensic analysis and the guidelines for publication of population data requested by the journal. Our institution passed the blind test (QC) offered by the YHRD and the US Y-STR database and has been certified. The Y-STR population data are being submitted to both the YHRD (www.yhrd.org) and the US Y-STR database (www.usystrdatabase.org). 6. Conclusion In conclusion, the data support that for the 17 Y STR loci in common between the Prototype PowerPlex1 Y23 System and AmpFlSTR1 YfilerTM kit allele discordance is rare for the three major US population groups (i.e. African Americans, Caucasians, and Hispanics) and three Native Alaskan populations (i.e., Inupiat, Yupik, and Athabaskan). A few novel duplications and null alleles were observed at the additional six Y STR loci. Typing samples with different primer sets and comparing the results serve as part of the validity testing of these kits for obtaining reliable results. Because of the different formats of different kits some nomenclature differences can arise and software should be developed to accommodate nomenclature differences to reduce false exclusions if Y STR data are used for database searches. Lastly, the six new Y STRs combined approach the PD of the 17 loci in the AmpFlSTR1 YfilerTM kit. Thus, the additional Y STR loci should provide increased power for differentiating male individuals. Acknowledgments We would like to thank Michelle Collins and Orin Dym at the Department of Public Safety, Scientific Crime Detection Laboratory in Alaska for their help in procuring Native Alaskan samples. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fsigen.2012.06.005. References [1] C. Leibelt, B. Budowle, P. Collins, Y. Daoudi, T. Moretti, G. Nunn, D. Reeder, R. Roby, Identification of a D8S1179 primer binding site mutation and the validation of a primer designed to recover null alleles, Forensic Sci. Int. 133 (2003) 220–227. [2] M. Heinrich, M. Mu˝ ller, S. Rand, B. Brinkmann, C. Hohoff, Allelic drop-out in the STR system ACTBP2 (SE33) as a result of mutations in the primer binding region, Int. J. Legal Med. 118 (2004) 361–363. [3] D.Y. Wang, R.L. Green, R.E. Lagace´, N.J. Oldroyd, L.K. Hennessy, J.J. Mulero, Identification and secondary structure analysis of a region affecting electrophoretic mobility of the STR locus SE33, Forensic Sci. Int. Genet. 6 (2012) 310–316.

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