Standards and Guidelines - Y-chromosome Short Tandem Repeat (Y-STR) Interpretation Guidelines - January 2009
January 2009 - Volume 11 - Number 1
Y-chromosome Short Tandem Repeat (Y-STR) Interpretation Guidelines
Scientific Working Group on DNA Analysis Methods (SWGDAM)
The interpretation of the results of casework is a matter of professional judgment and expertise. Not every situation can or should be covered by a preset rule. It is important that the laboratory develop and implement written guidelines for the interpretation of analytical results. This document provides a framework for the laboratory to develop Y-chromosome short tandem repeat (Y-STR) interpretation guidelines. The laboratory’s interpretation guidelines should be based upon validation studies, data from the literature, instrumentation used, and/or casework experience.
Y-STR data may augment autosomal STR results. In some circumstances, Y-STR data might be the only data that can be obtained. It is important to note that a Y-STR haplotype is shared by males from the same paternal lineage. This fact must be taken into account when drawing conclusions.
1.1. The laboratory should develop criteria to determine whether the results are of sufficient intensity/quality for interpretation purposes using methods appropriate for the detection platform. These criteria should be determined by evaluating data generated by the laboratory.
1.1.1. When quantitative results (e.g., peak amplitude) are used to evaluate STR profiles, the results should be examined to determine if they meet the laboratory’s empirically defined analytical and interpretational threshold(s).
22.214.171.124. The analytical thresholds are defined as the minimum and maximum intensity thresholds between which data are reliable for use in allele designations.
126.96.36.199. The interpretational threshold, the minimum intensity threshold that an allele must meet to be included in a biostatistical calculation, should be defined.
1.2. The laboratory should develop criteria to evaluate internal lane size standards and/or allelic ladders.
1.3. Controls are required to assess analytical procedures.
1.3.1. The laboratory should establish criteria for the evaluation of the controls used in the testing, for example, reagent blank controls, amplification blank controls, female positive controls, and male positive controls.
1.3.2. The laboratory should develop criteria for the interpretation and documentation of results based on evaluation of the controls.
1.4. A laboratory using Y-chromosome STR multiplexes that contain redundant loci should establish criteria regarding the concordance of such data.
2.1. The laboratory should establish criteria to assign allele designations to appropriate peaks or bands.
2.1.1. Locus Designation: The laboratory should establish criteria to address locus assignment for alleles.
2.1.2. Allele Designation: The laboratory should designate alleles in concordance with the recommendations of the DNA Commission of the International Society of Forensic Genetics.
188.8.131.52. Whenever possible, allele designation should be based operationally on the number of repeat units contained within the allele and by comparison to an allelic ladder.
184.108.40.206. The designation of alleles containing an incomplete repeat motif (i.e., an off-ladder allele falling within the range spanned by the ladder alleles) should include the number of complete repeats and, separated by a decimal point, the number of base pairs in the incomplete repeat (e.g., DYS385 15.2 allele).
220.127.116.11. If an allele falls above the largest or below the smallest allele of the allelic ladder, the allele should be designated as either greater than (>) or less than (<) the respective ladder allele, or when appropriate, extrapolation can be used.
18.104.22.168. For some duplicated loci, the alleles cannot be assigned unequivocally to a defined genetic locus. In concordance with recommendations of the DNA Commission of the International Society of Forensic Genetics, the results should be treated as a genotype (e.g., DYS385 11–14).
2.2. Artifacts can occur and should be noted. These may include, but are not limited to, the following: pull-up, stutter, incomplete nontemplate nucleotide addition, and nonspecific female DNA amplification.
3.1. The interpretation guidelines should include criteria to determine if an observed peak is a true allele as outlined in Section 2.1. The laboratory should define conditions in which the data would lead to the conclusion that the source of the male DNA is from either an apparent single male or two or more males of different paternal lineages. This may be accomplished by an examination of the number of alleles at each locus and the peak-height (or band-intensity) ratios at those loci that exhibit locus duplication such as DYS385.
3.1.1. Apparent Single Male Contributor: A sample may be considered to represent a single male haplotype when the observed number of alleles at each locus is one and the signal intensity ratio of alleles at a duplicated locus is consistent with a profile from a single contributor. All loci should be evaluated in making this determination. It should be noted that individuals have been typed who exhibit multiple locus duplications at loci other than DYS385.
3.1.2. Mixtures with Major/Minor Male Contributors: A sample may be considered to consist of a mixture of major and minor male contributors if a distinct contrast in signal intensity exists among the alleles. All loci should be evaluated in making this determination.
3.1.3. Mixtures with a Known Male Contributor(s): In some cases, when one of the male contributors (e.g., the victim) is known, the genetic profile of the unknown male contributor may be inferred. Depending on the profiles in the specific instance, this can be accomplished by subtracting the contribution of the known male donor from the mixed profile.
3.1.4. Mixtures with Indistinguishable Male Contributors: When major or minor male contributors cannot be distinguished because of similarity in signal intensities or the presence of shared or masked alleles, individual males may still be included or excluded as possible contributors.
3.2. The laboratory should have guidelines for the interpretation of partial profiles (i.e., profiles with fewer loci than tested) that may arise from degraded or limited-quantity DNA or from the presence of polymerase chain reaction (PCR) inhibitors. Occasionally, deletion of a portion of the Y-chromosome or a primer-binding site mutation can result in the failure to detect one or more Y-STR loci.
4.1. The laboratory should prepare guidelines for formulating conclusions resulting from comparisons of evidentiary samples and known reference samples.
4.1.1. General categories of conclusions are inclusionmatch, exclusion or nonmatch, inconclusive or uninterpretable, and no results. or
4.1.2. Comparison of haplotypes cannot distinguish between males from the same paternal lineage; therefore, inclusions need to be qualified.
5.1. Y-STR loci are located on the nonrecombining part of the Y-chromosome and, therefore, should be considered linked as a single locus. A Y-STR database must consist of haplotype frequencies rather than allele frequencies. The source of the population database(s) used should be documented. Relevant population(s) for which the frequency will be estimated should be identified. A consolidated U.S. Y-STR database (http://usystrdatabase.org) has been established and should be used for population frequency estimation. A number of other Y-STR haplotype frequency databases exist online. (See available listing on the NIST [National Institute of Standards and Technology] STRBase Web site at http://www.cstl.nist.gov/biotech/strbase/y_strs.htm.)
5.2. In reporting matches, haplotype searches of the population database should be conducted using all loci for which results were obtained from the evidentiary sample. In cases where less information is obtained from the known sample, only those loci for which results were obtained from both the known and evidentiary samples should be used in the population database search.
5.3. The basis for the haplotype frequency estimation is the counting method. The application of a confidence interval corrects for database size and sampling variation. Reporting a haplotype count without a confidence interval is acceptable as a factual statement regarding observations in the database.
5.3.1. If a confidence interval is applied, the following example calculation could be used:
22.214.171.124. The haplotype has not been previously observed in the database:
The formula for calculating the upper 95 percent confidence limit in this case would be
where n is the size of the database.
126.96.36.199. The haplotype has been observed in the database:
The formula for calculating the upper 95 percent confidence limit in this case would be
where p is x/n, n = database size, and x = the number of observations of the haplotype in the database.
5.4. For Y-STR mixtures that cannot be deconvoluted, calculations may be performed for the probability of exclusion and likelihood ratios.
5.5. If both autosomal and Y-STR data are collected on a sample, the product rule may be used to combine the autosomal STR genotype match probability and Y-STR haplotype frequency information.
5.6. It is recognized that population substructure exists for Y-STR haplotypes. Studies with current population databases have shown that the FST values are very small for most populations. Thus the use of the counting method that incorporates the upper-bound estimate of the count proportion offers an appropriate and conservative statistical approach to evaluating the probative value of a match.
6. References/Suggested Readings
Balding, D. J. Weight-of-Evidence for Forensic DNA Profiles. John Wiley & Sons, Hoboken, New Jersey, 2005, pp. 99–101.
Ballantyne, J., Fatolitis, L., and Roewer, L. Creating and managing effective Y-STR databases, Profiles in DNA (2006) 9(2)10–13. Also available: http://www.promega.com/profiles/902/ProfilesinDNA_902_10.pdf.
Buckleton, J., Walsh, S., and Harbison, S. Nonautosomal forensic markers. In: Forensic DNA Evidence Interpretation. J. Buckleton, C. M. Triggs, and S. J. Walsh, Eds. CRC Press, Boca Raton, Florida, 2004, pp. 299–331.
Budowle, B., Adamowicz, M., Aranda, X. G. et al. Twelve short tandem repeat loci Y chromosome haplotypes: Genetic analysis of populations residing in North America, Forensic Science International (2005) 150:1–15.
Budowle, B., Jianye, G., and Chakraborty, R. Basic principles for estimating the rarity of Y-STR haplotypes derived from forensic evidence. In: Proceedings of the Eighteenth International Symposium on Human Identification, 2007. Available: http://www.promega.com/geneticidproc/ussymp18proc/oralpresentations/Budowle.pdf.
Budowle, B., Sinha, S. K., Lee, H. S., and Chakraborty, R. Utility of Y-chromosome short tandem repeat haplotypes in forensic applications, Forensic Science Review (2003) 15:153–162.
Butler, J. M. Recent developments in Y-short tandem repeat and Y-single nucleotide polymorphism analysis, Forensic Science Review (2003) 15:91–111. Available: http://www.cstl.nist.gov/biotech/strbase/pub_pres/Butler2003b.pdf.
Butler, J. M. Y Chromosome DNA Testing. In: Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers. 2nd ed. J. M. Butler, Ed. Elsevier Academic Press, New York, 2005, pp. 201–239.
Butler, J. M., Decker, A. E., Kline, M. C., and Vallone, P. M. Chromosomal duplications along the Y-chromosome and their potential impact on Y-STR interpretation, Journal of Forensic Sciences (2005) 50:853–859. Available: http://www.cstl.nist.gov/strbase/pub_pres/ButlerJFS_Y-STRduplication.pdf.
DNA Advisory Board. Quality assurance standards for forensic DNA typing laboratories, Forensic Science Communications http://www.fbi.gov/about-us/lab/forensic-science-communications/fsc/july2000/codispre.htm. [Online]. (July 2000). Available:
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Gill, P., Brenner, C., Brinkmann, B. et al. DNA Commission of the International Society of Forensic Genetics: Recommendations on forensic analysis using Y-chromosome STRs, International Journal of Legal Medicine (2001) 114:305–9 and Forensic Science International (2001) 124:5–10.
Gusmão, L., Butler, J. M., Carracedo, A. et al. DNA Commission of the International Society of Forensic Genetics (ISFG): An update of the recommendations on the use of Y-STRs in forensic analysis, Forensic Science International (2006) 157:187–197 and International Journal of Legal Medicine (2006) 120:191–200.
Gusmão, L., Sánchez-Diz, P., Calafell, F. et al. Mutation rates at Y chromosome specific microsatellites, Human Mutation (2005) 26:520–528.
Hammer, M. F., Chamberlain, V. F., Stover, D. et al. Population structure of Y chromosome SNP haplogroups in the United States and forensic implications for constructing Y chromosome STR databases, Forensic Science International (2006) 164:45–55.
Kayser, M., Brauer, S., Schädlich, H. et al. Y chromosome STR haplotypes and the genetic structure of U.S. populations of African, European and Hispanic ancestry, Genome Research (2003) 13:624–634.
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Krenke, B. E., Viculis, L., Richard, M. et al. Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex, Forensic Science International (2005) 148:1–14.
Mulero, J. J., Chang, C. W., Calandro, L. M. et al. Development and validation of the AmpFSTR® Yfiler™ PCR amplification kit: A male specific, single amplification 17 Y-STR multiplex system, Journal of Forensic Sciences (2006) 51:64–75.
National Research Council, Committee on DNA Forensic Science. The Evaluation of Forensic DNA Evidence. 2nd ed. National Academies Press, Washington, D.C., 1996.
Prinz, M., Ishii, A., Coleman, A., Baum, H. J., and Shaler R. C. Validation and casework application of a Y chromosome specific STR multiplex, Forensic Science International (2001) 120:177–188.
Schoske, R., Vallone, P. M., Kline, M. C., Redman, J. W., and Butler, J. M. High-throughput Y-STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays, Forensic Science International (2004) 139:107–121. Available: http://www.cstl.nist.gov/div831/strbase/pub_pres/Schoske2004.pdf.
Scientific Working Group on DNA Analysis Methods (SWGDAM). Short Tandem Repeat (STR) Interpretation Guidelines, Forensic Science Communications [Online]. (July 2000). Available: http://www.fbi.gov/about-us/lab/forensic-science-communications/fsc/july2000/index.htm/strig.htm.
Sinha, S. K., Budowle, B., Chakraborty, R. et al. Utility of the Y-STR typing systems Y-PLEX™ 6 and Y-PLEX™ 5 in forensic casework and 11 Y-STR haplotype database for three major population groups in the United States, Journal of Forensic Sciences (2004) 49:691–700.
Walsh, B., Redd, A. J., Hammer, M. F. Joint match probabilities for Y chromosomal and autosomal markers, Forensic Science International (2008) 174:234–238.