Uses of the NIST 26plex STR assay for human identity testing

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
• 3. Results and discussion
• Conflict of interest statement
• Acknowledgments
• References
• Copyright

Abstract 

Ongoing work at the U.S. National Institute of Standards and Technology has focused on the characterization of 26 autosomal STR loci for human identity testing. These 26 loci are in addition to the existing 13 U.S. core loci and those found in PowerPlex16 and Identifiler commercial STR typing kits. The amplification of the 26 loci has been optimized for degraded extracts in unique miniplex panels and also for reference samples as a single reaction 26plex assay. A study has been performed comparing genotypes obtained with the 26plex primers to those with miniplex panels for allele drop out and concordance. The forensic utility of the 26plex assay was evaluated for situations where additional loci are beneficial. The utility of this large multiplex was also tested in a case involving DNA extracted from degraded bone samples. The 26plex can serve as a low-cost assay (compared to commercially available kits) useful for both sorting comingled remains and providing additional markers for increased statistical support for samples that require “non-trio” family references for human identification.

Keywords: STR, Additional loci, Kinship, Paternity, Multiplex, PCR

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1. Introduction 

Typing STR loci in addition to the common 13–15 loci found in commercial kits can help provide assistance in complex kinship testing. The NIST 26plex STR assay is well suited for typing reference samples where ∼0.5ng of pristine DNA is available for analysis [1]. A strategy can be employed in which miniSTR panels are used for typing degraded extracts and the 26plex assay is used for typing for pristine family reference samples [2]. Depending on the typing success of the miniSTR panels, the resulting information from overlapping loci is then used for likelihood ratio calculations and kinship determination.

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2. Materials and methods 

Three samples were tested for kinship analysis. Two reference samples K1a (brother) and K1b (maternal niece) and a degraded unknown (Q1) were genotyped for 33 STR loci. The two reference samples (K1a and K1b) were typed with Identifiler™ (15 STR loci) (Applied Biosystems, Foster City, CA) and the NIST 26plex assay (25 STR loci). The degraded unknown sample (Q1) was typed with MiniFiler™ (8 STR loci) (Applied Biosystems) and 10 panels of NIST miniplexes with loci that overlapped with the 26plex assay [2]. PCR conditions and data analysis were carried out as previously described [1], [2], [3], [4], [5]. Likelihood ratio (LR) calculations were determined for the full sibling and avuncular relationships. Allele frequencies for the loci used in LR calculations were taken from STRBase (http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm). The LR calculations were performed with the Identifiler loci using DNA-View (Charles Brenner, Oakland, CA) or for the non-CODIS markers using an excel spreadsheet for each relationship (kindly provided by Steven Myers and John Tonkyn, California DOJ) utilizing the formulas within Brenner and Weir (2003) [6].

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3. Results and discussion 

Fig. 1 shows the hypothesized kinship relationships in question. In the case of K1a (brother) the eight Minifiler loci resulted in a LR of ∼300. The 25 additional loci resulted in an increased LR of ∼3.7 million. The combination of all 33 overlapping loci resulted in a combined LR of 1.1 billion. In the case of the K1b (maternal niece), the eight Minifiler loci resulted in a LR of ∼2. The 25 additional loci resulted in a LR of ∼3.6. The combination of all 33 overlapping loci resulted in a combined LR of ∼7. In the case of the full sibling relationship, the additional 25 STR loci significantly increased the LR and thereby the confidence in the kinship determination. However, for the more distant avuncular relationship (with an expected 1/4 sharing of alleles), the extra 25 STR loci only provided a minimal increase to the calculated LR. This is not unexpected given the amount of allele sharing expected from relatives outside of the traditional paternity trios. Additional information from lineage makers (e.g. mitochondrial control region sequences) may be needed to further examine the relationship.

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• Fig. 1. 

  Schematic of hypothesized relationships and resulting likelihood calculations.

The NIST 26plex assay provides a single tube PCR for typing 25 STR loci plus Amelogenin. These 25 loci are not found in U.S. commercial DNA STR typing kits or linked to the CODIS loci. The additional loci contained in the 26plex assay can supplement commercial kits when 15 STR are not sufficient for a reliable kinship determination.

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Conflict of interest statement 

This work was funded in part through interagency agreement 2008-DN-R-121 between the National Institute of Justice and the NIST Office of Law Enforcements. Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the U.S. Department of Justice or Department of Defense. Commercial equipment, instruments, and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.

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Acknowledgments 

The authors wish to thank Suzanne Barritt, Layla Shahmirzadi, Chris Johnson, Kimberly Sturk, Brion Smith and Lou Finelli (U.S. Armed Forces DNA Identification Laboratory), Bruce Weir and Mary-Claire King (University of Washington), and Steven Myers California Department of Justice.

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References 

1. Hill CR, Butler JM, Vallone PM. A new 26plex assay for use in human identity testing. J. Forensic Sci. 2009;54:1008–1015
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2. Hill CR, Kline MC, Coble MD, Butler JM. Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples. J. Forensic Sci. 2008;53:73–80
   • View In Article
   • CrossRef
3. Butler JM, Hill CR, Decker AE, Kline MC, Reid TM, Vallone PM. New autosomal and Y-chromosome STR loci: characterization and potential uses. In: Proceedings of the Eighteenth International Symposium on Human Identification. 2007;
   • View In Article
4. Applied Biosystems, 2001, AmpFlSTR^® Identifiler™ PCR Amplification Kit User's Manual, Foster City, CA, P/N 4323291.
   • View In Article
5. Applied Biosystems, 2007, AmpFlSTR^® MiniFiler™ PCR Amplification Kit User's Manual, Foster City, CA, Part Number 4374618 Rev. B.
   • View In Article
6. Brenner CH, Weir BS. Issues and strategies in the DNA identification of World Trade Center victims. Theor. Popul. Biol. 2003;63:173–178
   • View In Article
   • MEDLINE
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