A Y‐Chromosomal Haplotype with Two Short Tandem Repeat Mutations*

Abstract:  The male‐specific Y‐chromosomal short tandem repeat (STR) is a useful tool in forensic casework. The Y haplotype comprised of 16 loci, which is amplified simultaneously by AmpFlSTR^® Yfiler^TM PCR kit and provides strong exculpatory evidence in individual identification. We reported a rare Y‐STR profile with a null allele at the DYS448 locus and an off‐ladder allele at the DYS456 locus, when genotyping material from a vaginal swab in an alleged rape case. Sequence analysis revealed that the DYS448 null allele was a true type of null allele because of a total deletion of 11 upstream repeats and 9 bp of the N₄₂ region, and there were numerous primer binding site mutations as well. The amplicon of the DYS456 locus was a small 92‐bp fragment that was off‐ladder, and sequencing analysis showed that there were only 10 repeats (AGAT)₁₀. This Y chromosome haplotype that was comprised of two variations provided helpful evidence for personal identification.     

Subtyping of D20S85 STR alleles by single-strand conformation polymorphism (SSCP) analysis

During a population study of STR locus D20S85, we discovered two types of sequence variations by direct sequencing of the alleles: two transitions each of G to A and A to G occur in the 5′ flanking region in the individuals possessing allele 6 and some of those possessing allele 7 [1]. Using single-strand conformation polymorphism (SSCP) analysis, we were able to distinguish two subtypes of allele 7 from each other. This analysis method enables rapid screening for STR alleles of the same length with different sequences, and should find application to other complex STR loci because of the practical advantage of simplicity in comparison to sequencing.     

Hypervariable locus of the 3'-flanking region of the neurotensin receptor gene: an effective region for personal identification in forensic practice

We examined the complex short tandem repeat (STR) locus at the 3'-flanking region of the neurotensin receptor (NTR) gene. The polymorphism of this locus was first reported as a simple tetranucleotide repeat variation by Le et al., but it also offers a surprisingly informative variation, that permits reliable individual identification by two complementary strategies: fluorescent-labelled polymerase chain reaction (PCR)/electrophoresis and direct sequencing of the PCR products. We determined the alleles in 203 Japanese by fluorescent-labelled PCR/electrophoresis. Determination was based on their length with a reliability of +/-1 bp, and the frequency of each allele was very low. Sequencing analysis further grouped these alleles in detail. Sequencing demonstrated that the locus varied by six repetitive units and three insertion/deletion positions of nucleotide fragments. We detected multiple alleles having different structures even in the same allele length. We found structural differences in homozygous alleles having the same base pair size. We also determined that apparently homozygous alleles were heterozygous from sequencing electropherograms showing an overlap of nucleotides or +/-1 bp difference. These results indicate that this locus is structurally hypervariable in addition to having allelic length variations, promising a great advance in individual identification in forensic practice.     

Characterization of a novel dimorphism in the 5' flanking region of the short tandem repeat (STR) locus,c-fes/fps (FES)

The FES short tandem repeat (STR) locus contains seven to 14 repeats of the tetranucleotide sequence ATTT. A novel 10 base pair dimorphism in the 5' flanking region of the FES locus was characterized in four broad populations: African-American, Hispanic, Caucasian, and Asian. The absence of the 10 base pair sequence, or (-) allele, was closely linked to FES STR alleles with 10 or fewer repeats. The presence of the 10 base pair sequence, or (+) allele, was closely linked to FES STR alleles with 12 or more repeats. The (-) and (+) alleles occurred equally often in FES STR allele 11. The nucleotide sequence (5'-GGCTGTTTTG-3') of the (+) allele, located 179 base pairs upstream of the FES STR, was determined to be consistent within and among the four populations. Statistical and sequence analysis confirmed the linkage between the two polymorphic sites. The results indicate that the exclusion rate of the FES locus is increased, above that for the STR alone, when both polymorphic characteristics are considered.     

The single most polymorphic STR Locus: SE33 performance in U.S. populations

The STR locus SE33 (ACTBP2) located on chromosome 6 (6q14) is arguably the most polymorphic marker examined thus far by the forensic community with a heterozygosity of >0.95 in some populations. Three different primer sets were utilized in this study in order to assess the possibilities of primer binding site mutations. Population variation was measured in 460 U.S. Caucasian, 445 African American, 336 Hispanic, and 202 Asian samples along with mutation rates from almost 400 father–son pairs. In addition, the 10 genomic DNA components in NIST Standard Reference Material SRM 2391b were sequenced and found to exhibit a variety of additional base changes, insertions, and deletions outside of the SE33 repeat region.     

Genetic variation and population genetic data of the short tandem repeat locus D8S320

The locus D8S320 is an STR system first described in 1993 as a simple (AAAG) repeat. Sequencing data revealed that the D8S320 locus is a complex STR system consisting of (AAAG)- and (AAAC)-repeat units. A total of 22 different alleles were found in a population survey of 210 unrelated individuals from the Rhine area with frequencies ranging from <0.01 to 0.198. The population data revealed the existence of variant alleles differing by 1 and 2bp from the consensus allele. Due to the complex repeat structure consisting of three variable regions and one constant region, electrophoresis under denaturing conditions is strongly recommended. The statistical values were calculated to be 0.89 (observed heterozygosity rate), 0.96 (discrimination index) and 0.71 (mean exclusion chance). No deviation from Hardy-Weinberg equilibrium (HWE) was observed.     

Additional sequence characterization of NIST SRM 2391c: PCR-Based DNA Profiling Standard

The NIST Standard Reference Material (SRM) 2391c: PCR-Based DNA Profiling Standard was designed for use in the standardization of forensic and paternity quality assurance procedures for fragment-based typing short tandem repeat (STR) alleles generated by the polymerase chain reaction (PCR). Certified genotypes of the 6 components A–F were assigned for 24 autosomal and 17 Y-STR markers plus Amelogenin using concordance testing between commercial kits. Selected Sanger sequencing characterization was performed for the alleles of 11 STR markers when only one PCR primer set was available for fragment-based typing. The goal is to characterize the remaining 30 STR loci in components A–C by Sanger sequencing methods for the STR repeat regions and adjacent flanking regions. Additional characterization of the SRM is intended to support the emerging interest in next-generation sequencing technologies for forensic typing applications. Sanger methods have detected underlying polymorphisms (sequence, insertion-deletion, variation in complex motifs) typically not detected by fragment-based typing. The sequenced regions include the commercial or known PCR binding sites commonly implemented in fragment-based typing.     

Characterization of deletions in the DYS385 flanking region and null alleles associated with AZFc microdeletions in Koreans

Eight DYS385 allele size discrepancies and six DYS448 null types were detected among 708 Korean men when results of three in-house multiplex short tandem repeat (STR) systems were compared. The systems included both ordinary and reduced size amplicons. Sequence analysis revealed deletion mutations at two sites upstream of the DYS385 core repeats and deletion of the entire DYS448 locus. At DYS385, allele size differences were one or two repeats and were dependent on the primer set used for polymerase chain reaction (PCR) amplification. Location of the primer target sequence in a flanking region of the STR, distal or proximal to the deletion, determined allele size. Two widely used commercial kits amplify DYS385 so as to include the mutable sites. Arrangement analysis of sequence tagged sites demonstrated that the deletion patterns at DYS448 (and DYS464) were associated with arrangements of the azoospermia factor c gene (AZFc). The DYS448 deletion appears relatively frequent in Asians.     

荧光标记STR复合扩增检验系统的研究

本研究以化学合成的荧光标记引物建立了STR复合扩增体系,体系I包括4个STR基因座和牙釉蛋白基因,体系Ⅱ包括4个STR基因座,体系Ⅲ包括3个STR基因座.制备了体系I各基因座的等位基因标准对照,测定了各等位基因长度.对代表性等位基因进行了序列测定,确定了等位基因重复单位的重复数目及测量长度与等位基因命名的对应关系,对各基因座等位基因进行了标准命名.建立了复合扩增检验系统等位基因自动分析命名的模板.三个复合扩增检验体系的累积随机匹配概率为8.3×10-3,为遗传学分析建立了实用的检验系统.     

Characterization of the variant allele 9.2 of Penta D locus

DNA profiles of forensic cases of Córdoba Province, Argentina, typed by PowerPlex 16 kit (Promega), have shown in the Penta D locus few samples with a variant allele migrating as an off ladder between alleles 9 and 10. In order to determine the molecular basis of the new variant allele, three samples were subject to polymerase chain reaction amplification of the Penta D locus by monoplex, and were further purified and sequenced. The sequence analysis revealed that the off ladder allele has ten repeats motifs AAAGA as allele 10, with three nucleotides (TAA) deletion in the 3' flanking region, 128 nucleotides after the last repeat. Therefore, the variant allele could be explained by a deletion of allele 10, and was designated 9.2. Mse I digestion assay allows to corroborate allele 9.2 without sequencing. A population study in Córdoba Province indicates that allele 9.2 of Penta D locus has a frequency of 0.0063.     

Make it "SNPPY" - Updates to SRM 2391d: PCR-Based DNA Profiling Standard

Standard Reference Material (SRM) 2391d: PCR-Based DNA Profiling Standard was released to the forensic community in 2019. Next Generation Sequencing (NGS) was used as the primary method of certification, where certified values were assigned when a high coverage sequence string was available for a marker. Using NGS to assign values has allowed for additional marker sets beyond short tandem repeat (STR) loci, including single nucleotide polymorphisms (SNPs) and mitochondrial DNA (mtDNA) whole genome sequences, to be included in the Certificate of Analysis (COA). Since the 2019 release, several commercial NGS panels have become available including the Verogen ForenSeq mtDNA Control Region, mtDNA Whole Genome, MainstAY, and Kintelligence Kits. In addition, three community Ion AmpliSeq panels from Thermo Fisher (MH-74 Plex, VISAGE, and Y-SNP) are now available. While the mtDNA whole genome sequence for the components are already included and no new STR markers are introduced by MainstAY, the other recently released panels allow for the inclusion of > 11,000 additional SNPs (e.g., identity, ancestry, phenotype, kinship, and X- and Y-SNPs) and 74 microhaplotypes to the COA for SRM 2391d in an update completed by fall of 2022.     

Unleashing novel STRS via characterization of genome in a bottle reference samples

The higher level of multiplexing possible with current sequencing technologies encourages adoption of additional STR loci to aid in mixture interpretation [1]. However, characterization of these loci and orientation on the human genome is vital for interlaboratory comparability and databasing. Currently, when a laboratory publishes population data from a locus not previously characterized for forensic use, there is no robust way to verify the locus designation, repeat region format, and fidelity of target. To address this, we have evaluated short- and long-read sequence data generated for reference materials included in the Genome in a Bottle Consortium (GIAB) [2] with the goal of reporting STR sequences for loci which may be of interest to the forensic community. Initially, we have analyzed GIAB data using Marshfield sets of primers (published in [3]), targeting over 600 microsatellite loci with STRaitRazor 3.0 [4]. In the future, this approach can be expanded to include other loci of interest. High-confidence STR sequence data will be made publicly available via GenBank record creation within the STRSeq BioProject [5]. As the cell lines represented in GIAB reference materials are available for purchase, this STR dataset represents a robust method for researchers to confirm targeted loci.     

Discordance at D3S1358 locus involving SGM Plus™ and the European new generation multiplex kits

During the course of routine database sample analysis in the Israel Police DNA database, an off-ladder D3S1358 allele, calculated to be >22.1, extending into the adjacent vWA locus was observed using Applied Biosystems SGM Plus™ kit.To verify the size of this D3S1358 long allele and to ensure it was not part of a trialle pattern in the neighboring locus, the sample was amplified using three of the European new generation STR multiplex kits: NGM^TM (Applied Biosystem), Powerplex™ ESX and ESI (Promega). The results of these amplifications determined the variant to be a 22 allele. Subsequent sequencing confirmed this designation and revealed a nucleotide polymorphism. Ten additional SGM Plus™ profiled samples with D3S1358 alleles larger than 19, were re-analyzed using NGM^TM and Powerplex™ ESX which also showed discordance in the calculated results between original SGM Plus™ designations and those obtained with the European new generation multiplexes.     

A proposal for standardization in forensic canine DNA typing: allele nomenclature of six canine-specific STR loci

In this study a proposal for the allele nomenclature of six polymorphic short tandem repeat (STR) loci (PEZ3, PEZ6, PEZ8, PEZ10, FHC2161, and FHC2328) for canine genotyping (Canis lupus familiaris) is presented. The nomenclature is based on the sequence data of the polymorphic region of the microsatellite markers as recommended by the DNA commission of the International Society of Forensic Haemogenetics (ISFH) in 1994 for human DNA typing. To cover commonly and rarely occurring alleles, a selection of homozygous and heterozygous animals were analyzed and subjected to sequence studies. The alleles consisted of simple tri- and tetra-nucleotide repeat patterns as well as compound and highly complex repeat patterns. Several alleles revealing the same fragment size but different repeat structures were found. The allele designation described here was adopted to the number of repeats, including all variable regions within the amplified fragment. In a second step the most commonly occurring alleles were added to an allelic ladder for each marker allowing a reliable typing of all alleles differing in size. A total number of 142 unrelated dogs from surrounding municipal animal homes, private households, and canines in police duty were analyzed. The data were added to a population database providing allele frequencies for each marker.     

A highly polymorphic STR locus in Cannabis sativa

We report on the first short tandem repeat (STR) locus to be isolated from the plant Cannabis sativa. The STR locus, isolated by a hybrid-capture enrichment procedure, was found to contain a simple sequence repeat motif of 6 bp. This 6 bp repeat motif showed no variation in repeat length but with minor variations in repeat unit sequences. The data show the locus to be highly polymorphic with the number of repeat units ranging from 3 to 40 in 108 screened samples. The observed heterozygosity was approximately 87.04%. The forward and reverse primers (CS1F and CS1R) produced no PCR products in cross-reaction study from 20 species of plants, including highly related species such as Humulus japonicus and Nicotiana tabacum. This hexanucleotide repeat DNA locus could be used to identify cannabis samples and predict their genetic relationship as the test is specific to C. sativa and is highly reproducible.     

Real-time forensic DNA analysis at a crime scene using a portable microchip analyzer

An integrated lab-on-a-chip system has been developed and successfully utilized for real-time forensic short tandem repeat (STR) analysis. The microdevice comprises a 160-nL polymerase chain reaction reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a co-injector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A 9-plex autosomal STR typing system consisting of amelogenin and eight combined DNA index system (CODIS) core STR loci has been constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples are obtained in 2 h and 30 min with ≤0.8 bp allele typing accuracy. The minimal amount of DNA required for a complete DNA profile is 100 copies. To critically evaluate the capabilities of our portable microsystem as well as its compatibility with crime scene investigation processes, real-time STR analyses were carried out at a mock crime scene prepared by the Palm Beach County Sheriff's Office (PBSO). Blood stain sample collection, DNA extraction, and STR analyses on the portable microsystem were conducted in the field, and a successful “mock” CODIS hit was generated on the suspect's sample within 6 h. This demonstration of on-site STR analysis establishes the feasibility of real-time DNA typing to identify the contributor of probative biological evidence at a crime scene and for real-time human identification.     

Allele frequencies of six miniSTR loci in the population of Northern Portugal

A possible approach to try to recover information from degraded DNA is to reduce the size of the PCR products by designing primers that bind as close as possible to the STR repeat region, known as miniSTRs. Allele frequencies and forensic parameters for the six miniSTRs loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045 were investigated in a sample group consisting of 228 anonymous apparently healthy unrelated individuals living in North of Portugal. The results show that all loci were in Hardy–Weinberg equilibrium. The combined power of discrimination and power of exclusion for the six loci were 0.99999 and 0.9789, respectively. All but one (D4S2364) loci showed a moderate degree of polymorphism (observed heterozygosity >0.6). The allele sizes ranged between 66 and 118 bp in our population, which is beneficial for typing degraded samples than those of a commercial STR kit.     

Typing Y chromosome STR haplotypes using redesigned primers

A panel of Y-specific STR loci, including DYS19, DYS389, DYS390, DYS391, DYS392, and DYS393 was analyzed using horizontal nondenaturing polyacrylamide gel electrophoresis with a discontinuous buffer system (horizontal disk-PAGE). In order to obtain correct results for the larger DYS389 and DYS392 alleles, it was necessary to design new primers that bind closer to the repeat region and lead to a significant reduction of the amplified fragment size. Using the modified primer sets the horizontal disk-PAGE results were consistent with a nondenaturing approach using fluorescent primers and a 377 automated sequencer. The modified procedure also amplifies the second repeat stretch at the duplicated DYS389 locus as a single fragment, which results in an immediate allele identification. The results indicate that horizontal disk-PAGE with silverstaining is a simple approach to type the recommended Y-specific STR markers.     

A DYS438 null allele observed in two generations of a large family

Typing of Y-chromosomal STR loci using the AmpFISTR^® Yfiler kit showed a DNA profile lacking the DYS438 allele in an Austrian Caucasoid brother pair.Buccal swabs were collected from additional males of two generations and different branches of the family. All family members investigated did not show a DYS438 allele in their Yfiler profile. Using the Powerplex^® Y system an allele with dramatically reduced peak height was amplified. Subsequent sequencing of the DYS438 locus exhibited a transition upstream of the repetitive region.