STRs vs. SNPs: thoughts on the future of forensic DNA testing
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Forensic Sci Med Pathol (2007) 3:200–205 DOI 10.1007/s12024-007-0018-1 ORIGINAL PAPER STRs vs. SNPs: thoughts on the future of forensic DNA testing John M. Butler Æ Michael D. Coble Æ Peter M. Vallone Accepted: 4 January 2007 / Published online: 12 September 2007 Ó Humana Press Inc. 2007 Abstract Largely due to technological progress coming material, we believe that STRs rather than SNPs will from the Human Genome and International HapMap fulfill the dominant role in human identity testing for the Projects, the issue has been raised in recent years within foreseeable future. However, SNPs may play a useful role the forensic DNA typing community of the potential for in specialized applications such as mitochondrial DNA single nucleotide polymorphism (SNP) markers as possi- (mtDNA) testing, Y-SNPs as lineage markers, ancestry ble replacements of the currently used short tandem informative markers (AIMs), the prediction of phenotypic repeat (STR) loci. Our human identity testing project traits, and other potential niche forensic casework team at the U.S. National Institute of Standards and applications. Technology (NIST) has explored numerous SNP and STR loci and assays as well as developing miniSTRs for Keywords DNA DNA typing DNA profiling degraded DNA samples. Based on their power of dis- Short tandem repeat Single nucleotide polymorphism crimination, use in deciphering mixture components, and MiniSTR STR SNP mtDNA ability to be combined in multiplex assays in order to recover information from low amounts of biological Introduction Official disclaimer: Contribution of the U.S. National Institute of Standards and Technology. Not subject to copyright. Points of view in Both length and sequence genetic variation exist in human this document are those of the authors and do not necessarily populations. These forms of variation enable forensic DNA represent the official position or policies of the U.S. Department of testing because many different alleles can exist in non- Justice. Certain commercial equipment, instruments and materials are coding regions of the genome. When information from identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a multiple unlinked genetic markers is combined, high recommendation or endorsement by the National Institute of powers of discrimination are possible. Length variation, in Standards and Technology nor does it imply that any of the materials, the form of short tandem repeat (STR) markers, has instruments, or equipment identified are necessarily the best available become the primary means for forensic DNA profiling over for the purpose. This work was funded in part by the National Institute of Justice through interagency agreement 2003-IJ-R-029 with the the past decade [1]. Large national DNA databases now NIST Office of Law Enforcement Standards. exist containing millions of STR profiles based on a few core STR markers [2, 3]. J. M. Butler (&) M. D. Coble P. M. Vallone While current STR systems and DNA databases are National Institute of Standards and Technology, Biochemical working well, the question often posed is ‘‘what does the Science Division, 100 Bureau Drive, Mail Stop 8311, Building 227, Room A243, Gaithersburg, MD 20899, USA future hold for forensic DNA testing?’’ Will another set of e-mail: john.butler@nist.gov genetic markers, such as single nucleotide polymorphisms (SNPs), replace the core STR loci already so effectively in Present Address: use? These questions were addressed in 2000 by the M. D. Coble Armed Forces DNA Identification Laboratory, Research Section, Research and Development Working Group of the National 1413 Research Blvd., Bldg 101, Rockville, MD 20850, USA Commission on the Future of DNA Evidence [4] and will
Forensic Sci Med Pathol (2007) 3:200–205 201 be considered here briefly based on the current state of the Significant SNP disadvantages science with STRs and SNPs. For a number of years, largely due to technology pro- Several significant disadvantages exist with SNP markers gress coming from the Human Genome and International when considered as a possible replacement for currently HapMap Projects [5], the primary potential replacement for used STR loci with the top two being the number of loci STRs has usually been proposed to be SNPs, which are needed and the inability to easily decipher mixtures. First, sequence variants that occur on average every several because SNPs are not as polymorphic as STRs, more SNPs hundred bases throughout the human genome [6]. Abun- are required to reach equivalent powers of discrimination dant SNP loci have been characterized and studied in or random match probabilities. Numbers on the order of various human populations [7]. 40–60 SNPs have been suggested in order to approximate Most articles to date discussing the potential applica- the power of 13–15 STR loci as are commonly in use today tions of SNPs in forensic DNA testing have focused on [10, 12, 19]. technology reviews [8, 9] or the number of markers needed Remember that 15 STRs can be routinely amplified to generate equivalent powers of discrimination in single simultaneously in a single multiplex amplification reaction source samples [10–12]. Recently work with improving the from minimal amounts (e.g., 500 pg) of DNA template level of multiplex amplification has been addressed [13, using commercially available kits such as PowerPlex 16 14], as has the selection of optimal SNP loci for use in and Identifiler. While multiplex PCR amplification of such forensic panels [15]. However, direct comparisons between a large number of SNPs (e.g., *50) has only recently been SNPs and STRs suggest that SNPs are not ready to replace demonstrated in a research setting [14], routine production STRs as the workhorse of forensic DNA identification and commercialization of robust assays containing upwards markers. of 100 oligonucleotide PCR primers will not be trivial. Likewise, the expense of examining more loci will be higher. Potential SNP advantages Perhaps more importantly data interpretation becomes increasingly difficult with more loci and amplification The primary reason provided for considering SNPs with products. Issues with locus drop out will become more forensic applications centers around the fact that a higher significant when three to five times more loci are involved recovery of information from degraded DNA samples is in comparison to traditional STR typing. In addition, assays theoretically possible since a smaller target region is nee- with a larger number of loci are more sensitive to the ded. Only a single nucleotide needs to be measured with quantity and integrity of the input DNA template particu- SNP markers instead of an array of nucleotides—some- larly when trying to amplify limited DNA materials. times hundreds of nucleotides in length—as with STRs Current SNP typing for use in HapMap population (Fig. 1). While this argument has been made for many studies (e.g., 7) or other clinical genetics projects involve years, a recent direct comparison for SNPs and STRs found attempting to type many thousands of SNP loci in a rela- that STR markers, when shortened to miniSTRs, perform tively small number of samples. If a few dozen or even quite well on degraded DNA material [13]. A wide variety hundreds of loci fail to produce a result on a sample, these of STR loci exist, and ones with moderate allele ranges and data are excluded from the final analysis or further attempts small amplicon sizes can work effectively with compro- are made using a replenishable supply of DNA. This type mised DNA samples. Primer redesign to create smaller of data loss when attempting to perform a direct compar- amplicons with core STR loci [16] and a number of new ison between a suspect and evidence is undesirable or even STR loci [17] has been described recently. unacceptable under the current paradigm of sample Another advantage of SNPs is that they possess muta- matching performed with STR typing on only a dozen or so tion rates approximately 100 thousand times lower than loci. Will investigators or the courts care that a fraction of STRs (10–8 vs. 10–3). Thus, theoretically SNPs, being more the SNP loci attempted failed to produce a result? With stable in terms of inheritance, could aid parentage testing in limited amounts of starting material in many situations, some cases or kinship analysis such as is performed with there may not be opportunities to repeat the testing in an identifying mass disaster victims [18]. However, Amorim effort to recover the lost loci. Furthermore, the loci missing and Pereira [19] note some unexpected drawbacks of using on reference samples may be different from those that SNPs in forensic kinship investigations based on simula- failed on the evidentiary material leaving even less of an tions. They predicted that a battery of 45 SNPs would overlap of successfully typed loci for comparison purposes. produce a higher frequency of cases where statistical evi- When attempting to analyze a greater number of genetic dence would be inconclusive when applied to routine loci, there will be an increased complexity of data to be paternity investigations [19]. examined. Depending on the SNP detection platform, the
202 Forensic Sci Med Pathol (2007) 3:200–205 Fig. 1 Comparison of (A) (Biallelic) STRs and (B) SNPs in terms of Conventional STR SNP the number of possible allele miniSTR combinations and relative size C of the target region 7 repeats 8 repeats T 9 repeats 10 repeats 11 repeats 12 repeats Target region (single nucleotide 13 repeats polymorphism) Target region (short tandem repeat) Alleles Genotypes (3 possible genotypes) C CC T TT CT Alleles Genotypes (28 possible genotypes) 7 7,7 8 7,8 8,8 9 7,9 8,9 9,9 10 7,10 8,10 9,10 10,10 11 7,11 8,11 9,11 10,11 11,11 12 7,12 8,12 9,12 10,12 11,12 12,12 13 7,13 8,13 9,13 10,13 11,13 12,13 13,13 data to be interpreted will vary. With so many loci being exist for SNP typing [8, 9, 21, 22]. The various chemistries typed, software interpretation will become more reliant on involved are far from being standardized. Without con- expert computer systems as the primary form of data sensus throughout the community on the SNP markers to analysis. More loci mean more peak signals and the be employed or the detection platform(s) to be utilized, potential for more artifacts. Thus, detailed data analysis SNP typing cannot gain a foothold over the dominance of will become more tedious and practically impossible the widely used STR typing systems. without validated expert systems. Although the argument has been made that data interpretation will be easier with SNPs because they do not possess stutter products or Likely future role of SNPs microvariants, in a certain sense these biological amplifi- cation artifacts provide greater confidence in results—i.e., However, SNPs may play a useful role in niche applica- that a measured peak with a stutter product is truly an STR tions such as mitochondrial DNA (mtDNA), Y-SNPs, allele rather than a spike or a fluorescent dye artifact. ancestry informative markers (AIMs), predicting pheno- However, from our point of view, the most significant typic traits, and other potential forensic case work disadvantage of SNP typing is that the limited number of applications. Coding region SNPs can fulfill a useful role alleles (typically two) for each SNP locus limits or prevents for separating common HV1/HV2 mitochondrial DNA reliable mixture interpretation. A major advantage with types [23, 24], and assays have been developed to reliably STRs in a forensic setting is that many possible alleles exist examine mtDNA coding region SNP variation [25, 26]. providing the possibility that the multiple contributors to a While Y-SNPs have limited utility for individualizing a mixture will have distinguishable (non-overlapping) sample, they may, depending on the population(s) of alleles. Figure 2 shows SNP and STR typing data obtained interest, be helpful in aiding estimations of ethnic origin on the same mixed DNA sample. While the STR results [27, 28]. clearly suggest more than one contributor based on the Predicting ancestry [29, 30] or phenotypic characteris- number of alleles present at multiple loci, an analyst would tics, such as red hair color [31] or eye color [32], is another find it difficult to determine that a mixture is present based role that SNPs may play in the future where the amount of on the six SNP loci shown. sample is not limited. In these cases, SNP typing could help Another challenge preventing routine application of provide investigators with information about a perpetrator SNPs today is that multiple possible detection platforms based solely on the biological evidence left at the crime
Forensic Sci Med Pathol (2007) 3:200–205 203 Fig. 2 Mixture detection on the (A) SNP typing results same samples with (A) SNPs SNP SNP SNP SNP SNP SNP and (B) STRs. The top panel in Locus 1 Locus 2 Locus 3 Locus 4 Locus 5 Locus 6 each section contains a mixture possessing as one of its Mixture TT components the same DNA that CC is shown as a single source in C T CT the bottom panel. Six different T C T C SNP loci (from a total of 70 SNPs evaluated; see [20]) are CC TT Single Source TT T shown in (A). Note that only CC C SNP locus 2 in the top panel of C T (A) has an allele imbalance suggesting that a possible second contributor is present. On the other hand in the top panel of (B), all 5 STR loci (B) STR typing results shown from the green dye channel of the Identifiler kit Mixture contain three alleles making mixture detection much easier Single Source scene. Research in these areas is on-going and not yet platform/chemistry for SNPs that is robust enough for ready for routine use. Thus, it is important to stress that forensic use (at least as good as STRs) has not been fully while we do not think autosomal SNPs will replace auto- developed. somal STRs anytime soon, the utility of various SNP assays should still be evaluated. It will be valuable to understand which SNP markers Summary may be useful when/if the technology platforms catch up to the level acceptable for forensic usage. Since SNP markers The overall points made in this article are summed up in are abundant, a larger pool of potential markers is available Table 1. While automation of SNP detection has improved for testing. The majority of SNPs are bi-allelic so a search significantly in recent years enabling millions of SNPs to be for ‘most informative’ loci is not required (conversely a examined on hundreds of samples in a relatively short period useful STR should have multiple well-populated allelic of time (e.g., 7), due to the large number of loci that must be states). For human identification purposes, loci with low co-amplified and the inability to easily decipher mixtures, we FST values and a heterozygosity of [40% are generally do not feel that SNPs stand on the horizon as future markers adequate for forensic usage [15]. The abundance of SNPs (i.e., replacing STRs) for widespread use in forensic DNA means that ‘poor’ markers can be thrown out since the testing. That being said, there are and will likely be important information content is essentially equivalent between loci (this would be done in the initial selection phase of a SNP panel). Table 1 A simple summary comparing the characteristics of STRs It is important to keep in mind that an infrastructure and SNPs exists that supports STR typing. Databases with core STR Characteristics STRs/miniSTRs SNPs loci contain millions of profiles and are still being popu- Success with degraded DNA + + lated. More than 10 years of expertise in STR typing is Power of discrimination (without + – present in many forensic laboratories. A question that significant multiplexing) should be asked is, ‘‘Is it worth the time and cost to convert Mixture detection/interpretation + – over to a new marker/technology?’’ The main proposed Other applications: benefits of SNPs are the potential for use on degraded Ethnicity estimation, physical traits, etc. – + samples and possible rapid—high throughout analysis. Commercial testing kits available + – However, it appears that miniSTRs complete well in the Consistent platform/assay for analysis + – arena of degraded samples [13], and a high throughput
204 Forensic Sci Med Pathol (2007) 3:200–205 niche roles for SNP typing. We agree with Gill et al. [33] that development of new DNA typing systems. Electrophoresis autosomal SNPs will likely supplement STR results rather 1999;20:1682–96. 11. Krawczak M. Informativity assessment for biallelic single than supplant them due to the immense investment already nucleotide polymorphisms. Electrophoresis 1999;20:1676–81. made in national DNA databases. 12. Gill P. An assessment of the utility of single nucleotide poly- morphisms (SNPs) for forensic purposes. Int J Legal Med 2001;114:204–10. 13. Dixon LA, Dobbins AE, Pulker HK, Butler JM, Vallone PM, Educational message Coble MD, Parson W, Berger B, Grubwieser P, Mogensen HS, Morling N, Nielsen K, Sanchez JJ, Petkovski E, Carracedo A, 1. The two primary advantages for SNPs include (a) Sanchez-Diz P, Ramos-Luis E, Brion M, Irwin JA, Just RS, potential ability to work well on degraded DNA Loreille O, Parsons TJ, Syndercombe-Court, Schmitter H, Stradmann-Bellinghausen B, Bender K, Gill P. Analysis of arti- because a small target region can be amplified and (b) ficially degraded DNA using STRs and SNPs-results of a lower mutation rates compared to STRs, which could collaborative European (EDNAP) exercise. Forensic Sci Int aid kinship testing. 2006;164(1):33–44. 2. Significant disadvantages for SNPs include needing 14. Sanchez JJ, Phillips C, Borsting C, Balogh K, Bogus M, Fon- devila M, Harrison CD, Musgrave-Brown E, Salas A, 40–60 loci to obtain equivalent match probabilities as Syndercombe-Court D, Schneider PM, Carracedo A, Morling N. 13–15 STRs commonly used today and the greater A multiplex assay with 52 single nucleotide polymorphisms for difficulty with mixture interpretation due to a limited human identification. 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