John M. Butler

Genetics and Genomics of Core Short Tandem Repeat Loci Used in Human Identity Testing

ABSTRACT: Over the past decade, the human identity testing community has settled on a set of core short tandem repeat (STR) loci that are widely used for DNA typing applications. A variety of commercial kits enable robust amplification of these core STR loci. A brief history is presented regarding the selection of core autosomal and Y‐chromosomal STR markers. The physical location of each STR locus in the human genome is delineated and allele ranges and variants observed in human populations are summarized as are mutation rates observed from parentage testing. Internet resources for additional information on core STR loci are reviewed. Additional topics are also discussed, including potential linkage of STR loci to genetic disease‐causing genes, probabilistic predictions of sample ethnicity, and desirable characteristics for additional STR loci that may be added in the future to the current core loci. These core STR loci, which form the basis for DNA databases worldwide, will continue to play an important role in forensic science for many years to come.

AutoDimer: a screening tool for primer-dimer and hairpin structures

The ability to select short DNA oligonucleotide sequences capable of binding solely to their intended target is of great importance in developing nucleic acid based detection technologies. Applications such as multiplex PCR rely on primers binding to unique regions in a genome. Competing side reactions with other primer pairs or template DNA decrease PCR efficiency: Freely available primer design software such as Primer3 screens for potential hairpin and primer-dimer interactions while selecting a single primer pair. The development of multiplex PCR assays (in the range of 5 to 20 loci) requires the screening of all primer pairs for potential cross-reactivity. However, a logistical problem results due to the number of total number of comparisons required. Comparing the primer set for a 10-plex assay (20 total primer sequences) results in 210 primer-primer combinations that must be screened. The ability to screen sets of candidate oligomers rapidly for potential cross-reactivity reduces overall assay devlelopment time. Here we report the application of a familiar sliding algorithm for comparing two strands of DNA in an overlapping fashion. The algorithm has been employed in a software package wherein the user can compare multiple sequences in a single computational run. After the screening is completed, a score is assigned to potential duplex interactions exceeding a user-defined threshold. Additional criteria of predicted melting temperature (Tm) and free energy of melting (deltaG) are included for further ranking. Sodium counterion and total stand concentrations can be adjusted for the Tm and deltaG calculations. The predicted interactions are saved in a text file for further evaluation.

Characterization of New MiniSTR Loci to Aid Analysis of Degraded DNA

A number of studies have demonstrated that successful analysis of degraded DNA specimens from mass disasters or forensic evidence improves with smaller sized polymerase chain reaction (PCR) products. We have scanned the literature for new STR loci, unlinked from the CODIS markers, which can generate amplicons less than 125 bp in size and would therefore be helpful in testing degraded DNA samples. New PCR primers were designed and tested for the STR loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434, and D22S1045, arranged into two miniSTR triplexes. All loci show a moderate degree of polymorphism among 474 U.S. population samples tested and were reliable and sensitive to at least 100 pg of DNA template under controlled laboratory conditions and pristine DNA samples. The utility of these new loci were confirmed in comparing the success of the miniSTR assays for typing degraded bone samples while partial profiles were observed with the majority of the samples using a commercial STR kit.

Publication of population data for forensic purposes

In 2000 a new policy concerning the publication of population genetic data was set up in Forensic Science International [1] with the introduction of a new section entitled ‘‘Announcement of population data’’. The idea was to facilitate the publication of this type of data since the use of reliable allele or haplotype frequency estimates of the polymorphisms is a requirement in most countries, both in forensic and in paternity cases. Announcements of population data consisted in short communications under a fixed format, avoiding the repetition of superfluous information (i.e., materials and methods) and concentrating the message on the key information needed for the use of genetic data for forensic and population genetics. In our opinion, this type of paper completely fulfilled the aims of the editors and, even more importantly, has made an essential contribution to the dissemination of common standards all over the world. In addition they have motivated forensic practitioners (especially in countries with little development in forensic genetics) to introduce themselves in forensic research. With the launch of the new journal, we decided to continue the same policy as a first step but keeping in mind that we have to move forward to increase the quality of the journal and to avoid having a journal exclusively devoted to announcements of population genetic data. The number of population genetic papers from the very beginning has continuously increased, representing now more than 60% of the submissions to the journal. Therefore, it is time to raise the threshold regarding the acceptance of this type of publication but taking into account the importance of the dissemination of standards and the motivation that this type of research represents for some groups and countries. For this reason, we have decided to move to a next step and to introduce a new section on Forensic Population Genetics in the journal. Manuscripts with population genetic content can be submitted to this section at http://www.ees.elsevier.com/fsigen/ using three types of formats: Forensic Population Genetics – Original papers: in this section full length papers on relevant population genetics issues of forensic interest will be considered for publication. The data should be original, the population genetic analysis must be of the highest quality and the data should have forensic relevance beyond the scope of simply reporting allele or haplotype frequencies. Forensic Population Genetics – Short communications: understanding that both the quality of population data and the relevance of results are crucial, short communications will have the format of the former ‘‘Announcements of population data’’ with some changes (see below) in order to guarantee their quality.

STRBase: a short tandem repeat DNA database for the human identity testing community

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/++ +strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes.

Short tandem repeat typing technologies used in human identity testing.

Short tandem repeat (STR) typing methods are widely used today for human identity testing applications including forensic DNA analysis. Following multiplex PCR amplification, DNA samples containing the length-variant STR alleles are typically separated by capillary electrophoresis and genotyped by comparison to an allelic ladder supplied with a commercial kit. This article offers a brief perspective on the technologies and issues involved in STR typing.

Forensic value of 14 novel STRs on the human Y chromosome.

We identified and characterized 14 novel short-tandem-repeats (STRs) on the Y chromosome and typed them in two samples, a globally diverse panel of 73 cell lines, and 148 individuals from a European-American population. These Y-STRs include eight tetranucleotide repeats (DYS449, DYS453, DYS454, DYS455, DYS456, DYS458, DYS459, and DYS464), five pentanucleotide repeats (DYS446, DYS447, DYS450, DYS452, and DYS463), and one hexanucleotide repeat (DYS448). Sequence data were obtained to designate a repeat number nomenclature. The gene diversities of an additional 22 Y-STRs, including the most commonly used in forensic databases, were directly compared in the cell line DNAs. Six of the 10 most polymorphic markers include the newly identified Y-STRs. Furthermore, these novel Y-STRs greatly improved the resolution of paternal lineages, above the level obtained with commonly used Y-STRs, in the European-American population.

A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome

The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) can help resolve individuals with an identical HV1/HV2 mitotype. A set of 11 SNPs selected for distinguishing individuals of the most common Caucasian HV1/HV2 mitotype were incorporated in an allele specific primer extension assay. The assay was optimized for multiplex detection of SNPs at positions 3010, 4793, 10211, 5004, 7028, 7202, 16519, 12858, 4580, 477 and 14470 in the mtGenome. Primers were designed to allow for simultaneous PCR amplification of 11 unique regions in the mtGenome and subsequent primer extension. By enzymatically incorporating fluorescently labeled dideoxynucleotides (ddNTPs) onto the 3’ end of the extension primer, detection can be accomplished with a capillary-based electrophoresis (CE) platform common in most forensic laboratories. The electrophoretic mobility for the extension primers was compared in denaturing POP4 and POP6 CE running buffers. Empirical adjustment of extension primer concentrations resulted in even signal intensity for the 11 loci probed. We demonstrate that the assay performs well for heteroplasmy and mixture detection, and for typical mtDNA casework samples with highly degraded DNA.

New guidelines for the publication of genetic population data.

In 2000 a new policy concerning the publication of population genetic data was set up in Forensic Science International [1] with the introduction of a new section entitled ‘‘Announcement of population data’’. Subsequently in 2010 [2] a new section on ‘‘Forensic Population Genetics’’ was introduced, and recommendations were redefined. FSI: Genetics is one of the few journals still considering population genetic data for publication and we strongly believe that this policy has contributed to the dissemination of common standards in the field all over the world and also to motivate labs and people to embark in research in the area of forensic genetics. For this reason it is our intention to continue with this policy, and recently an associate editor exclusively devoted to this topic was appointed to the journal. Despite having defined a more detailed procedure for acceptance, our journal is still receiving a massive number of submissions of varying quality in this area. Therefore it has become necessary to raise the threshold regarding the acceptance of this type of publication to ensure a high standard of published data. In addition we want to improve the submission, reviewing and publication procedures, and to correct some aspects that we have detected such as the obligation to meet ethical standards in the collection of samples including informed consent and approval by ethical committees. For this reason, we have decided to publish new guidelines for the publication of population genetic data in the journal.

Update of the guidelines for the publication of genetic population data

Due to the massive number of submissions of varying quality with population genetic data we decided one year ago to raise the threshold regarding the acceptance of this type of publications to ensure a high standard of published data and, therefore, we updated the FSI: Genetics 2010 guidelines [1] to a new set of recommendations [2]. In the 2013 guidelines in addition to some new requirements in the procedure and regarding ethical standards, we significantly increased the number of markers and samples required for submission to the journal of papers presenting population data alone, with no additional information on new methods or other forensically relevant findings. We have been working during last year with these new recommendations. During this period, we have received a number of critical comments from authors that we have considered and therefore we have decided to make some amendments in the requirements. The first change refers to the minimal number of autosomal STRs. The 2013 guidelines indicate that for data comprising autosomal STR genotypes only, 17 different autosomal STR loci are required as a minimum. This was based on the average number of STRs that most laboratories are routinely using – in most cases forensic laboratories are using at least two PCR multiplexes (commercial kits or homemade) on their routine work for a minimal number of 17 autosomal STR markers. However, this number would exclude laboratories and national and international compilation efforts that are working with just one kit, for some of the most commonly used kits. Whilst we certainly recognize the need to restrict the number of small data sets submitted we think that the forensic community would benefit from the publication of a large and nationally important data set such as the ones that are being generated in some countries. For this reason we have decided to reinstate the 2010 recommendations requiring 15 STRs only. Concerning X chromosome a minimum number of 12 STRs will be required and for the Y chromosome a minimum of 17 STRs will be required as well, taking into account that the core minimum haplotype (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385) [3,4] must be provided if it has not been previously analyzed in the same population sample. Collaborative efforts to produce large datasets are strongly encouraged and, therefore, the minimal number of markers should not be a limitation to the publication of large National or International collaborative databasing efforts when a significant number of laboratories and samples are involved. There are also changes in the requirements for the minimum number of samples. We maintain the threshold of 500 samples for