Analysis of Y-chromosome STR markers

Abstract

The methods and compositions provided herein relate to the discovery of 13 STRs that are markers present on the human Y chromosome that have unexpectedly high mutation rates when compared to the 173 other Y-STR markers known today, including those commonly used in forensics. In addition to theoretical expectations based on increased mutation rates, these 13 rapidly mutating (RM) Y-STRs have been shown to be suitable for distinguishing close male relatives, as well as more distant male relatives, and are better suited for such distinctions than the most commonly used Y-STRs in forensics. Our new RM-Y-STR panel, due to its unusual mutational properties as discovered herein, is expected to overcome the current dilemma of Y-chromosome analysis in forensic applications in many cases, ranging from current male pedigree identification to male individual identification. Embodiments of the invention include methods for allelic determination of rapidly mutating Y-STR markers, amplification primers for analyzing rapidly mutating Y-STR markers, allelic ladders for analyzing rapidly mutating Y-STR markers, and kits for analyzing rapidly mutating Y-STR markers.

Description

[0001] This application is a divisional application. The application date of the original application is September 10, 2010, the application number is 201080051273.9 (PCT / US2010 / 048542), and the name of the invention is “Analysis of Y-chromosome STR markers”.

[0002] This application claims priority to U.S. Provisional Application No. 61 / 241,778, filed September 11, 2009, U.S. Provisional Application No. 61 / 367,346, filed July 23, 2010, and U.S. Provisional Application No. 61 / 379,340, filed September 1, 2010. Application Nos. 61 / 241,778, 61 / 367,346, and 61 / 379,340 are incorporated herein by reference in their entirety for any purpose.

[0003] field

[0004] Embodiments of the subject invention are in the field of forensic analysis of DNA. background

[0005] The use of STR markers has become a standard tool in analyzing DNA found at crime scenes. In most cases, the use of autosomal STR markers is utilized, in part because of their high level of polymorphism in most populations. For example, in the United States, the standard 13 CODIS loci used to establish a suspect DNA database are autosomal STR markers. In many cases with mixed blood from male and female contributors, especially in rape cases, forensic investigators must analyze genetic markers present on the Y chromosome to identify the male component that is usually a criminal. This is because in such cases, autosomal STR markers are not informative due to, for example, spectral overlap between female victim DNA and male criminal DNA. Although there are technical possibilities for preferentially obtaining male DNA (i.e., differential cleavage), such techniques are often unsuccessful. Because female DNA lacks a Y chromosome, analysis of Y chromosome markers can be used for samples containing high levels of female DNA relative to male DNA in the sample. Therefore, analyzing Y chromosome DNA eliminates complex artifacts caused by excessive female-derived DNA.

[0006] The non-recombinant nature enables the use of Y-chromosome markers for male pedigree identification, i.e., groups of males that are paternally related and therefore share the same Y-STR haplotype, i.e., based on the Y-STR markers currently used in forensics. Male pedigree identification has become an extremely useful tool for exclusion of males in forensic genetics. However, in non-exclusionary (i.e., matching Y-STR profiles) cases, individual-based statements cannot be made based on existing Y-STR markers, since a male suspect and all his male relatives have the same probability of contributing to the crime scene sample. This is clearly a limitation in forensic applications where individual-based conclusions are desired. However, mutation events can occur in Y-STR markers. In principle, these mutations in Y-STR markers can enable investigators to distinguish between close male relatives, and also more distant males, provided that such mutations occur in a given pair of male relatives at a high enough frequency that they can be observed. Mutations in existing Y-STR markers are fairly rare events, occurring at about 0.1 to 0.4% (1-4 changes per Y-STR locus per thousand generational events). Therefore, even when a relatively large number of Y-STR markers are used (i.e., the 17 markers currently used for forensic applications), the probability of distinguishing male relatives is still extremely low. However, if enough Y-STR markers are available that mutate faster than the currently known Y-STRs, it is expected that close male relatives as well as distant male relatives can be distinguished based on Y-STR mutations to achieve the desired male individual identification in forensic applications.

[0007] The inventors have discovered a subset of 13 Y-STR markers that have significantly higher mutation rates than most Y-STR markers, including those that are widely used. This discovery is expected to revolutionize the use of the Y chromosome in forensic biology, which is different from previous methods of distinguishing male pedigrees. The discovery also provides a direction for the identification of male individuals. Therefore, by using one or more, by using two or more such rapidly mutating Y-STR markers (RM Y-STRs), the ability to distinguish between close and distant male relatives is significantly increased.

[0008] Overview

[0009] Certain embodiments of the invention include methods of identifying an individual by determining the alleles of at least two Y-STR markers selected from the group consisting of DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627. In some embodiments of the methods, the alleles can be identified by PCR. In some embodiments of the methods, the alleles can be identified by mass spectrometry. The PCR can be a multiplex PCR to co-amplify the at least two rapidly mutating Y-STR markers. Certain embodiments of the present invention include amplification primer pair sets including primers for amplifying at least 2 Y-STR markers selected from the group consisting of DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627. The primer set can co-amplify at least 2-13 rapidly mutating Y-STR markers. In certain embodiments, the primer set can co-amplify autosomal STR markers in addition to rapidly mutating Y-STR markers. In some embodiments, the autosomal STR can be selected from D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, THO1, TPOX, and CSF1PO. In some embodiments, the primers can be labeled with a fluorescent dye. Other embodiments provided are allelic ladder size standards for retrieving one or more alleles of a STR from at least two Y-STR markers selected from the group consisting of DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627. Other embodiments provided are kits for identifying alleles of at least two Y-chromosome STRS markers, wherein the markers are selected from the group consisting of DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627, the kit comprising primers for amplifying at least two rapidly mutating Y-STR markers, and an allelic ladder representing the selected markers.

[0010] Brief Description of the Figures and Schedules

[0011] Figure 1 . Mutation rates for 186 Y-STR markers established from father-son pair analysis. The distribution of 186 Y-STR markers was estimated based on their Bayesian-based mutation rates (with confidence intervals) by analyzing up to 1966 DNA-confirmed father-son pairs for each marker. The 13 rapidly-mutating (RM) Y-STR markers identified for further family / pedigree analysis are highlighted in red, and the 17 commonly used Yfiler Y-STRs are indicated in green. Multicopy Y-STRs are indicated by black inserted diamonds.

[0012] Figure 2 Correlation between the length of the longest homogeneous array or the total number of repeats within a locus and allele-specific mutation rates from 267 Y-STR loci. Although the number of repeats present within a locus can be used to predict mutability, the total number of repeats present within a locus has a higher predictive value.

[0013] Figure 3 . Relationship between total number of repeats and direction of mutation and mutation rate from 267 Y-STR loci. Repeat loss mutations (contraction) show an exponential relationship with total number of repeats, with loss rates increasing at loci with higher numbers of repeats. Repeat gain mutations (expansion) show a weak quadratic function, with gain rates having a peak at 20 total repeats.

[0014] Figure 4 . Differentiation of male relatives using 13 newly identified RM Y-STRs and 17 commonly used Yfiler Y-STRs. Results from analysis of 103 pairs from 80 male pedigrees for differentiation of male relatives based on the 13 RM Y-STRs (in red) and 17 Yfiler Y-STRs (in blue), sorted by the number of generations separating the pedigree members. Error bars represent 95% binomial confidence intervals. Note that these samples are independent of the father-son pairs originally used to establish the Y-STR mutation rates.

[0015] Table 1. Mutation rate estimates based on the posterior distribution (median and 95% confidence interval) of 186 Y-STR markers, from analysis of up to 1966 DNA-confirmed father-son pairs. -2Markers with median mutation rates (RM Y-STR group) are highlighted. Also included are the observed marker repeat structure, number of gains / losses, total mutations, and total number of father-to-son transmissions. PCR primers, PCR annealing temperatures, and loci used for genotyping are assigned to 54 multiplexes and 3 RM Y-STR multiplexes.

[0016] Table 2. Details of 924 mutations observed in 120 Y-STR markers based on a total of 352,999 meiotic transfers screened at 186 Y-STR markers. Where possible, the repeat structure of both the paternal and son alleles of the mutant Y-STR is given. In the case of multi-copy markers with multiple variable segments within the amplicon, the total repeat number or amplicon size is given in the absence of sequence information. The father's age at the son's birth is given as a reference for the individual pair.

[0017] Table 3. Comparison of 13 rapidly mutating RM Y-STRs and 17 Yfiler Y-STRs that distinguish male relatives by 1 or more mutations, according to the number of generations separating members of the same pedigree, by analyzing 103 pairs from 80 male pedigrees.

[0018] definition

[0019] A "mutation" in a Y-STR marker is a change in the length of the repeat region of the STR marker or a change in the length (i.e., number) of bases interspersed with the repeat units. For example, the addition of one or more repeat units is a mutation that results in the appearance of a new allele. In another example, the addition of a single base within a single repeat unit is also a mutation that results in the appearance of a new allele. Such changes can be the result of the addition or deletion of one or more repeat units (or fragments thereof). Such sequence changes can be readily detected by analytical methods capable of detecting variations in nucleic acid sequence length or nucleic acid base sequence.

[0020] As used herein, the term "rapidly mutating Y-STR markers" (RM Y-STRs) refers to the following 11 Y-STR markers: DYF387S1, DYF399S1, DYF404S1, DYS449, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627.

[0021] As used herein, the term "allelic ladder" refers to a standard size marker consisting of amplified alleles from a given STR locus or to a size standard that is equivalent in size (or electrophoretic mobility) to an amplified allele from a given STR locus. An allelic ladder may include size standards for one or more alleles of a given STR marker. An allelic ladder may include alleles from different STR markers. The size standards in an allelic ladder may be labeled with a detectable label, such as a fluorescent dye.

[0022] The term "Y-STR marker" as used herein refers to an STR marker present on the non-recombining portion of the human Y chromosome. Based on current knowledge, there are more than 250 such Y-STR markers. Y-STR markers are well known to those of ordinary skill in the art. Databases of Y-STR markers are publicly available, for example, at the websites www.usystrdatabase.org and www.yhrd.org.

[0023] As used herein, the term "STR" refers to a region of genomic DNA containing short repetitive sequence elements. The repeated sequence elements are not limited to, but are typically 3 to 7 base pairs in length. Each sequence element is repeated at least once within a STR and is referred to herein as a "repeat unit." The term STR also includes regions of genomic DNA in which more than a single repeat unit is repeated, either in tandem or with intervening bases, provided that at least one sequence is repeated at least twice in tandem.

[0024] The term "primer" as used herein refers to a single-stranded oligonucleotide or DNA fragment that hybridizes with the DNA strand of a locus in such a manner that the 3' end of the primer can serve as a site for polymerization and extension using a DNA polymerase. In addition to or in place of naturally occurring DNA, primers may also be DNA analogs, such as LNA, base analogs, and the like. A "primer pair" refers to two primers, including a primer 1 that hybridizes with a single strand at one end of a DNA sequence to be amplified, and a primer 2 that hybridizes with the other end on a complementary strand of the DNA sequence to be amplified. A "primer site" refers to a target DNA region to which a primer hybridizes.

[0025] As used herein, the indefinite article and "the" and similar references used herein should be understood to cover both the singular and the plural, unless the context indicates otherwise. Thus, when the indefinite article is used with the term "comprising" in a claim or specification, it may mean "one" but is also consistent with "one or more", "at least one", and "one or more than one". It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely", "only", etc., in connection with reciting claim elements, or for the use of a "negative" limitation.

[0026] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs.All patents, patent applications, published applications, papers and other publications mentioned herein are incorporated herein by reference in their entirety.If the definition and / or description set forth herein is contrary to or otherwise inconsistent with any definition set forth in the patents, patent applications, published applications and other publications incorporated herein by reference, the definition and / or description set forth herein will take precedence over the definition combined by reference.Citation to any publication is the disclosure prior to the filing date and should not be construed as admitting that the present invention will not be eligible to precede the publication due to prior invention.

[0027] Description of Certain Specific Embodiments

[0028] The applicants have determined the mutation rates of numerous Y-STRs by examining three areas: i) the lack of knowledge regarding Y-STR mutability based on the reasonably large number of loci required for evolutionary and genealogical applications, ii) the limited knowledge regarding the molecular basis of Y-STR mutability, and iii) the lack of Y-STRs for family differentiation in forensic, genealogical, and specific population applications.

[0029] Table 1 provides mutation rates and properties of 186 Y-STR markers in approximately 2000 DNA-confirmed father-son pairs. Mutation rate estimates are included, most of which are determined for the first time. Diversity and DNA sequence data generated for all loci were also evaluated to explore potential causes of Y-STR mutability. The most mutable Y-STRs identified were tested for suitability for distinguishing male relatives and their significance for the application of the Y-chromosome in forensic science, resulting in the identification of 13 rapidly mutating Y-STR (RM- Y-STR) markers.

[0030] The 13 Y-STR markers were found to have significantly higher mutation rates than the other 173 Y-STRs tested. These rapidly mutating markers are DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627. The mutation rates of these 13 RM-Y-STRs are well over 10 -2 , while all other 173 Y-STRs (94% of the loci tested) had a probability of being completely below 10 -2 (Usually 10 -3 and lower) mutation rate ( Figure 1 In particular, the locus-specific mutation rates for the 13 RM Y-STRs ranged from 0.0116 to 0.0744. In comparison, the 17 Y-STRs included in the AmpF / STR® YFiler™ PCR Amplification Kit (YFiler Kit, sold by Applied Biosystems / Life Technologies, Foster City, California USA, namely DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385 a / b * , DYS393, DYS391, DYS439, DYS635, DYS392, YGATA H4, DYS437, DYS438, DYS448) ranged from 0.0002 to 0.0065, which was recently established based on a large number of >135,000 meiotic transfers (Goedbloed et al. 2009). Thus, Applicants unexpectedly discovered that 13 RM-Y-STRs mutate faster (60-11 times) than the YFiler kit Y-STRs most commonly used for forensic applications today. The unexpectedly high mutation rates in these RM-Y-STR markers increase the likelihood of distinguishing between male members of the same paternal genetic lineage. The likelihood of distinguishing between members of the same male lineage is even greater when multiple rapidly mutating Y-STR markers are employed. Various embodiments of the invention provided herein include methods, reagents, and kits for determining the specific alleles of one or more, two or more, three or more, four or more, five or more, etc., rapidly mutating Y-STR markers of the invention in a given analytical sample.

[0031] Various methods are provided herein for determining the specific alleles of one or more rapidly mutating Y-STR markers. The specific alleles of rapidly mutating Y-STR markers can be determined using substantially the same methods and techniques used to determine the alleles of other types of STR markers. The methods and techniques can be readily modified by one skilled in the art to be applicable to the determination of the alleles of rapidly mutating Y-STR markers. Examples of such techniques include DNA sequencing and sequence-specific amplification techniques such as PCR in conjunction with detection techniques such as electrophoresis, mass spectrometry, and the like. In some embodiments, the PCR amplification products can be detected by fluorescent dyes conjugated to PCR amplification primers, such as those described in PCT Patent Application WO 2009 / 059049. The PCR amplification products can also be detected by other techniques, including but not limited to staining the amplification products such as silver staining, and the like.

[0032] The specific allele of a given rapidly mutating Y-STR marker can also be determined by any of a variety of widely available DNA sequencing techniques, such as Sanger sequencing, pyrosequencing, Maxim and Gilbert sequencing, and the like.

[0033] Numerous automated DNA sequencing technologies are commercially available, including applied Biosystems 3130, applied Biosystems 3100, Illumina Genome Analyzer, Applied Biosystems SOLiD system, Roche Genome Sequencer Fix system, etc.

[0034] DNA for analysis using the present methods and compositions can be obtained from a variety of sources. DNA can be obtained at crime scenes, such as semen recovered from rape victims. In addition, DNA for analysis can be obtained directly from male subjects to generate an allele information database (for subsequent analysis) or can be obtained from identified suspects.

[0035] The DNA used for analysis can be quantified and then subjected to allelic analysis, thereby providing more accurate allele calling. The amount of DNA in a sample can be determined using many techniques known to those skilled in the art, such as real-time PCR. Of interest is the quantification of Y chromosome DNA present in the analysis sample followed by allelic analysis of Y-chromosome STR markers, including rapidly mutating Y-chromosome STR markers. The autosomal DNA in the sample can also be quantified, thereby providing a method for determining the background amount of female DNA present in mixed samples, such as those recovered in rape cases.

[0036] Y chromosome haplotypes can be established by determining the specific alleles present on a variety of Y-STR markers. In general, the faster the Y-STR markers mutate, the greater the likelihood that male relatives can be distinguished based on Y-chromosome marker analysis. In some embodiments, the rapidly mutating Y-STR markers can be analyzed by a method using multiplex PCR. Multiplex PCR can amplify 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 of the rapidly mutating Y-STR markers. In some embodiments, multiplex PCR can co-amplify additional Y-STR markers that are not part of the rapidly mutating Y-STR marker set of the present invention. In some embodiments, multiplex PCR can provide for co-amplification of one or more autosomal STR markers, such as CODIS STR markers, D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, THO1, TPOX, and CSF1PO. For a detailed description of the development of multiplex PCR for STR analysis, see, among other places, PCT patent application WO 2009 / 059049 A1. In some embodiments, the PCR reaction is not multiplexed. The amplicons generated in the non-multiplex PCR reactions can be combined and then analyzed by an instrument, such as a fluorescent DNA fragment analyzer (e.g., an automated DNA sequencer) or a mass spectrometer. Mass spectrometry analysis of STR markers can be found in, among other places, U.S. Patent No. 6,090,558.

[0037] Other embodiments include PCR primer sets for co-amplifying at least two rapidly mutating Y-STR markers. Embodiments include PCR primer sets for co-amplifying 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 of the rapidly mutating Y-STR markers provided herein. In some embodiments, the PCR primer set may include primers for co-amplifying Y-STR markers that are not rapidly mutating Y-STR markers. In some embodiments, the PCR primer set may include PCR primers for co-amplifying STR markers present on autosomes.

[0038] Embodiments of the present invention also include an allelic ladder to aid in the identification of alleles of rapidly mutating Y-STR markers. The allelic ladder may include a set of size standards for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 of the rapidly mutating Y-STR markers. For each marker present in the allelic ladder, the allelic ladder may include a standard for one or more alleles. The allelic ladder may include size standards for all known alleles of a given rapidly mutating Y-STR marker, or any subset of known alleles. In some embodiments, the size standards in the allelic ladder may be labeled with one or more fluorescent dyes. In some embodiments, the allelic ladder may also include size standards for autosomal STR markers. In some embodiments, the allelic ladder may also include size standards for Y-STR markers that are not rapidly mutating Y-STR markers.

[0039] Other embodiments of the invention include kits for determining the alleles of two or more rapidly mutating Y-STR markers. Embodiments of the kits may include the amplification primer sets of the invention. In some embodiments, the kits may include one or more reagents for a nucleic acid amplification reaction. Examples of the reagents include, but are not limited to, DNA polymerases, dNTPs, buffers, nucleic acid purification reagents, and the like. In some embodiments, the kits may include an allelic ladder designed as a size standard for one or more rapidly mutating Y-STR marker alleles that are produced (or may be produced) by the amplification primers present in the kit. Thus, in some embodiments, the kits may include an allelic ladder specifically adapted for use in amplicons produced by using the kit primers in an amplification reaction. For example, a kit that includes primers for co-amplifying rapidly mutating Y-STR markers DYF387S1, DYF399S1, and DYF404S1 may also include an allelic ladder with size standards for various alleles of the rapidly mutating Y-STR markers DYF387S1, DYF399S1, and DYF404S1. The kit may contain primers for co-amplification of all 13 RM-Y-STRs and an allelic ladder with appropriate size standards as known to those skilled in the art. For a given STR marker, the component size standards of the allelic ladder may be labeled with a detectable label, such as a fluorescent dye, that is the same or different than the primers used to generate the actual allelic amplicons in the assay sample.

[0040] The present invention can be better understood by reference to the following embodiments comprising experimental data. The information is provided as examples and is not intended to limit the scope of the invention claimed. The examples and data proposed herein are published in K. Ballantyne et al. "Mutability of Y-Chromosomal Microsatellites: Rates, Characteristics, Molecular Bases and Forensic Implications" Am. J. Hum. Genet. 87: 341 -353 (September 10, 2010) and published online on September 2, 2010, each incorporated herein by reference. Example

[0041] sample

[0042] In addition to family or government documents, all father-son pairs used for mutation rate studies also confirm their paternity by molecular analysis using autosomal STR, Y-STR, HLA and RFLP genotyping and blood typing. The probability threshold for paternity included in this study was set to 99.9%. Samples were obtained from Berlin, Leipzig and Cologne in Germany and Warsaw and Wroclaw in Poland. Due to the low amount of DNA, whole genome amplification was performed on Leipzig samples using GenomiPhi DNA amplification kit (GE Healthcare, Little Chalfont, UK). WGA reactions were performed according to the manufacturer's recommendations, and the products were purified using Invisorb 96 Filter Microplates (Invitek GmbH, Berlin, Germany). An additional independent set of samples from male relatives (not used for the initial screening of male families or pedigrees for mutability, used to validate the values ​​of the identified rapidly mutating Y-STRs) came from the Greifswald, Kiel and Berlin regions in Germany, the Leuven region in Belgium, the Warsaw region in Poland, as well as Canada and central Germany, as described elsewhere12. All families / pedigrees were confirmed by the same method as for father-son pairs; pairs with complete genotypes for both rapidly mutating (RM) Y-STRs and Yfiler Y-STRs were considered for analysis, or in the case of partial genotypes, only those showing mutations at one or more loci were included. The use of all samples for the purposes of this study was in accordance with institutional regulations and was performed with informed consent.

[0043] Marker and genotyping protocols

[0044] Y-STR markers were selected primarily from previous studies that detailed a large number (167) of previously unknown Y-STRs 29, with the addition of 42 Y-STRs known at the start of the project. The focus was on single-copy Y-STR markers so that genotypic differences could be fully confirmed by DNA sequence analysis when identifying mutations. However, given our goal to find RM Y-STRs, we included some additional multi-copy Y-STRs, particularly those with high diversity (mutation confirmation by independent genotyping). A complete list of loci, primer sequences, and protocols can be found in Supplementary Data S1 online. Seventeen of the 186 Y-STRs were genotyped using a commercially available kit, the AmpF / STR Yfiler PCR Amplification Kit (Applied Biosystems), following the manufacturer's instructions. A full description of the protocols and markers can be found in (28). The remaining 169 Y-STRs were genotyped using 54 multiplex assays, each including 1 to 5 markers. PCR was performed using 3 different protocols, and details are provided in Supplementary Data S1. In addition, 13 Y-STRs identified as rapidly mutating (RM) Y-STRs during the study were genotyped using 3 multiplex assays in an independent sample set of male relatives. All PCRs were performed on a GeneAmp PCR System 9700 instrument (Applied Biosystems) at the Department of Forensic Molecular Biology, Erasmus MC Rotterdam. Fragment length analysis was performed using a 3130x / Genetic analyzer (Applied Biosystems) at Applied Biosystems, Foster City, USA. Genotyping of the generated profiles was performed using GeneMapper software (ID v 3.2, Applied Biosystems). Genotypic differences were identified using an in-house developed Microsoft Excel 2007 macro. All mutations in both father and son at the Y-STR loci were confirmed by DNA sequence analysis in Rotterdam as described in M. Goedbloed et al. (2009) Int. J. Legal. Med. 123, 471-482. Multicopy Y-STR loci with three or more alleles could not be sequenced, but mutations were confirmed by at least two independent fragment length analysis amplifications.

[0045] Statistical data analysis

[0046] As described in Goedbloed et al., the mutation rates of individual markers were estimated using a binomial hierarchical Bayesian model43 using Marcov Chain Monte Carlo (MCMC) Gibbs sampling as implemented in WinBUGS. In short, each mutation rate was considered to be a realization of the potential mutation rate of any Y-STR. In short, we considered the mutation rate of Y-STR i to be is derived from hyperparameters The estimated Y-STR mutation rate thus incorporates information provided by the observed data for that Y-STR (the number of mutations observed in all observed father-son pairs) as well as information about the mutation rate of "that Y-STR" estimated in the hyperparameters from all Y-STRs. In practice, this implies that a Y-STR with no observed mutations will show a smaller mutation rate (estimated by the posterior distribution) than other Y-STRs with a large number of observed mutations, but always different from zero.

[0047] The mutation rate of each Y-STR is coded in logit form and is assumed to follow a normal distribution with parameters to be estimated and As well as the specific mutation rates of each STR. Since only very limited data on the range of Y-STR mutation rates were available before our study, we assumed diffuse, uninformative prior distributions with respect to the hyperparameters. Specify an uninformative prior normal distribution (μ=0, =1 x10 "6 ) and for the parameter Specify with parameter α=1x10 -5 and β = 1x10 -5Prior diffusion γ distribution.When estimating the mutation rate of each locus, 3 MCMC chains were generated in parallel with Gibbs sampler (sampler), and each chain was run 100,000 times.After abandoning the initial 50,000 runs and carrying out 15 thinning (thinning), the mean, median and 95% credible interval (CI) were estimated according to 3 chains to reduce the amount of autocorrelation between adjacent simulations.The locus-specific differences of mutation rate between sampling populations (Cologne, Berlin, Leipzig, Warsaw and Wroclaw) were tested by permutation analysis (permutation analysis).The average mutation rate of each locus and each population was compared with the assumed permutated population (permutated population), wherein each father and son were randomly assigned to the population, and the original sample size was kept for each locus.The average mutation rate of the arrangement was greater than the number of times of the observed rate, and was used to obtain the single-tail p value in 100,000 iterations (iterations).The lack of significant differences between populations makes it possible to merge the mutation rate between populations.

[0048] To explore the mutation rates of the Yfiler and RM Y-STR groups rather than the mutation rates of individual markers within the groups, the total number of mutations observed between each father-son pair for each group was calculated given the number of Y-STRs analyzed. This parameter was then modeled according to a Bayesian paradigm with a Poisson distribution. A prior with a gamma distribution was used with a diffuse shape of 1 and a scale of 200, which implies a mutation rate with a mean of 0.005 and a variance of 40,000. The posterior distribution follows a conjugate gamma distribution with a shape of 1 + (total number of mutations) and a scale of 1 / (1 / (200 + total number of markers used)). To estimate the probability of observing at least one mutation in each group, 100,000 Monte Carlo replicates were performed using the gamma function of the R package 45 based on the estimated shape and scale of the posterior distribution of each group of Y-STRs.

[0049] For the RM Y-STR panel, the estimated median mutation rate was 0.0197 (95% CI 0.018 - 0.022), which is approximately 7 times higher than the median rate of 0.0028 (95% CI range 0.0023 to 0.0035) shown by the YFiler panel of 17 markers. Next, the probability of observing at least one mutation per Y-STR panel in a given father-son pair (reflecting the lowest standard for distinguishing male relatives) was estimated as 1 minus the probability of observing 0 mutations, which is estimated directly from the Poisson distribution: The probability of observing at least one mutation (k) within any YSTR panel in any given father-son pair is estimated directly from the Poisson distribution: , where N represents the number of markers and m represents the average mutation rate of the marker set sampled from the posterior distribution. Assuming that all Y-STRs in each group have been successfully genotyped and using the posterior estimates of the mutation rates of each group of markers, the probability of observing at least one mutation in the RM Y-STR group is 0.1952 (95% confidence interval of 0.177 to 0.21). This value is more than 4 times the value estimated by the YFiler group (0.047 (95% confidence interval of 0.038 to 0.057)), but the YFiler group includes an additional 6 markers relative to the RM Y-STR group. The molecular factors that determine the mutation rate were modeled using Poisson regression using an internally developed Matlab script (v7.6.0.324, The Mathworks, Inc., Natick, MA, USA). The mutation rate was modeled as a function of repeat length, sequence motif, locus complexity, and repeat length in base pairs (tri-, tetra-, penta-, or hexanucleotides) as:

[0050]

[0051] It is assumed Depending on the above factors, in the following form:

[0052]

[0053] Where L represents the length of the allele (number of repeats, the longest homologous array or the total locus), S represents the sequence motif (composed of the number of A, T, C or G nucleotides in the repeat sequence motif), C represents the complexity of the locus (in binary or quantitative form), V is the number of variable motifs present, R is the repeat length, and N is the copy number of the locus. A stepwise regression procedure was used with a probability of entry of <0.05 and a probability of removal of ≥0.10. For clarity, the method for defining and calculating the number of repeats within a locus and the complexity of the locus will be explained below.

[0054] Locus nomenclature was modeled according to Kayser et al., where at least 3 consecutive repeats of the same motif were required to define a given repeat segment as a locus, and any breaks of more than one base, but less than a complete unit, were classified as terminating the locus. Individual Y-STR loci contained between 1 and 5 repeat blocks, such as DYS612 with 5 blocks (CCT)5(CTT)1(TCT)4(CCT)1(TCT)19. If a locus contained more than one variable segment, and the repeat number could not be accurately assigned to all repeat segments for all individuals, the locus was removed from the regression analysis. If a change in repeat number was observed in any sequenced individual relative to the rest of the population, the segment was defined as variable.

[0055] Repeat number: directly calculate the number of repeats in the longest homologous array, and calculate the population mean of each locus. In addition, any additional repeats around the longest array are added to calculate the total number of repeats for each locus. In the example of DYS612 above, the longest array length is 19, and the total number of repeats is 30.

[0056] Repeat length: The length of the repeat motif in base pairs, which ranges from 3 to 6 (including tri-, tetra-, penta-, hexa- and heptanucleotide repeats).

[0057] Complexity: Two complexity statistics were calculated for each locus. First, a binary classification system was used, where loci with only one repeat segment (e.g., (GATA)10) were classified as simple, while any loci with two or more repeat segments (consisting of more than 3 consecutive repeats) (e.g., (GATA)10(CATA)3) were classified as complex. Second, the complexity formula of Kayser et al., as follows, provides more quantitative information: , where n is the total number of repeats in the locus, s i For the i The number of repetitions of the sequence motif, and For the i block Correlation and log-linear regression analyses were performed in SPSS v15.0 (SPSS Inc.), as were all mean comparison tests (using ANOVA, Mann-Whitney U and Kruskal Wallis).

[0058] Repeat length: The base pair length of the repeat motif, which ranges from 3 to 6 (including tri-, tetra-, penta-, hexa-, and heptanucleotide repeats).

[0059] Mutation rate of Y-STR markers

[0060] To define the expectation of a given RM Y-STR set to distinguish male relatives and compare this potential to the commonly used YFiler set, a Bayesian approach was applied to obtain the average mutation rate for each of the two Y-STR sets. For the STR set, the number of mutations observed in a father-son pair was modeled with the help of a Poisson distribution. A prior conjugate gamma distribution with a diffusion shape of 1 and a scale of 1 / 0.005 was used. A posterior distribution following a gamma distribution was obtained with a shape of 1 + total number of mutations and a scale of 1 / (1 / 0.005 + total number of markers used) and 100,000 Monte Carlo replicates were performed.

[0061] In addition, to test whether the new RM Y-STR set is practical and useful for distinguishing male relatives in independent samples, 107 pairs from 80 male pedigrees that were related between 1 and 20 generations within the pedigree were genotyped for both marker sets and the results were compared with those obtained from YFiler. Pedigrees are from the Greifswald and Kiel (N. von Wurmb-Schwark, V. Malyusz, E. Simeoni, E. Lignitz, M.Poetsch, For. Sci. Int. 159, 92-97 (2006) and Berlin (new to this study) regions in Germany, the Leuven region in Belgium (new to this study), the Warsaw region in Poland (new to this study), and from Canada C.Moreau, H. Vezina, V. Yotova, R. Hamon, P. de Kniff et al., Am. J. Phys.Anthropol. 139, 512-522 (2009), M. Vermeulen, A. Wollstein, K. van der Gaag,O. Lao, Y. Xue et al., For. Sci. Int. Genet., 3, 205-213 (2009) and Central Germany M. Kayser, M. Vermeulen, H. Knoblauch, H. Schuster, M. Krawczak, L. Roewer, For. Sci. Int. Genet. 1, 125-128 (2007)), as described elsewhere. All pedigrees were confirmed by DNA data (including autosomal STR, HLA and RFLP typing, Y-STR and Y-SNP typing and mtDNA sequencing between various pedigrees) and additionally by family or government file records. Only pairs with complete genotypes for both groups, or in the case of partial genotypes, pairs showing mutations at one or more loci were included in the calculations. Figure 2 Results are provided in . The RM Y-STR panel distinguished over 65% of pairs by at least 1 mutation, reflecting a 5-fold higher level of male relative discrimination than the YFiler panel (only 13%), which is similar to our statistical expectations for the initial father-son pair analysis. Within pedigrees, the RM Y-STR panel distinguished 60% of father-son pairs, 54% of brothers, and 87% of second cousins. If relatives were separated by more than 11 meioses, 100% of individuals were separated by 1 or more mutations using the RMY-STR panel. In contrast, the Y-filer panel did not distinguish father-son pairs and second cousins ​​in this dataset, and only distinguished 6% of brothers in this dataset.

[0062] By screening for mutations in each of 186 tri-, tetra-, penta-, and hexanucleotide Y-STR markers in up to 1966 DNA-confirmed father-son pairs by multiplexed fluorescence-based fragment length analysis, 352,999 meiotic transfers were directly observed (see Table 1 for technical details). To confirm mutations, all Y-STR genotype differences observed between father and his son were confirmed by DNA sequence analysis for single-copy and dual markers, or by dual fragment length genotyping analysis for multicopy Y-STRs (with more than 2 copies), where sequence analysis was non-informative. Overall, we identified 924 confirmed mutations in 120 (64.5%) of the 186 Y-STR markers studied (details of each observed mutation can be found in Supplementary Data S2). For 66 Y-STR markers, up to 1966 father-son pairs analyzed did not allow us to detect mutations due to the very low potential mutation rate. The large number of Y-STR markers used determined a Bayesian range of mutation rates that was estimated to be 3.81 x 10 per marker per generation based on the median of the posterior distribution. -4 (95% CI 1.38x10 -5 Up to 2.02x10 -3 ) to 7.73x10 -2 (6.51x10 -2 Up to 9.09x10 -2 )( Figure 1 , Table 1). The mutation rate of 91 Y-STR markers (48.9%) was about 10 -3 , and another 82 markers (44%) were about 10 -4 , and 13 (6.9%) were about 10 -2 Among all 186 Y-STR markers, the average mutation rate was 3.35x10 -3 (95% CI 1.79x10 -3 Up to 6.38x10 -3 ), the average rate for the 122 tetranucleotide repeats included as the largest repeat length subset of Y-STR markers in this article is 4.26x10 -3 (95% CI 2.38x10 -3 Up to 7.60x10 -3 ). It is worth noting that the mutation rate exceeds 1x10 -2 The 13 Y-STR markers that represent only 7% of the markers studied, which are referred to as "rapidly mutating Y-STRs" (RM Y-STRs), cover the majority of mutations observed in this study (462 of 924 (50%)).

[0063] Repeat number. Two estimates of the average repeat number were calculated for each Y-STR locus, i) the average repeat number in the longest homology array; and ii) the repeat number of the longest homology array plus any non-variable repeats in the nearest neighbours (according to rules previously determined for motif structure 29). Our regression analysis showed that although the repeat number in the longest homology array significantly affected the mutation rate (with higher repeat numbers increasing mutation rate (Wald χ 2 = 2.41 x 10 6 , p <0.0001 )), but including the nonvariable number of repeats around the array provided slightly more accurate information for the model (Wald χ 2 = 3.03 x 10 6 , p<0.0001, Figure 2 ). Estimate the effective size within the model, bias =0.798, indicating that the variance in the total number of repeats between loci accounts for approximately 78% of the total (effective + error) variance in the observed Y-STR mutation rates. In addition, a statistically significant exponential relationship (R 2 = 0.707, p = 6.84x10 -9 ). In addition, there is a strong relationship between the total number of repeats and the direction of mutation ( Figure 3 The longer allele showed an exponential and statistically significant trend toward repeat loss (contraction) (R 2 = 0.585, p = 8.27x10 -7 ), while the shorter alleles acquired duplications (expansions) significantly more frequently (R 2 = 0.238, p = 0.011). The expansion mutation rate has a quadratic distribution with a peak at approximately 19 repeats.

[0064] Distinguishing male relatives by RM Y-STR

[0065] We identified 13 rapidly mutating (RM) Y-STR markers (mutation rates >1x10 -2 ); DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627 ( Figure 1, Table 1). Four of the 13 RM Y-STR markers are multicopy systems (DYF387S1 is double copy, DYF399S1 is triple copy, DYF403S1 is quadruple copy, DYF404S1 is double copy, and DYS526 is double copy), while nine are single copy Y-STR markers (although six of these markers contain multiple Y-STR loci within a single amplicon, and only two, DYS570 and DYS576, are simple repeats, each with only one Y-STR locus). The 13 RM Y-STRs were combined into one group based on the assumption that if the RM Y-STRs were combined, close male relatives (even father-son pairs or brother pairs) could be distinguished by Y-STR mutations. In principle, one mutation in one of the 13 RM Y-STRs would be sufficient to distinguish individuals.

[0066] To define the statistical expectation of the RM Y-STR set for distinguishing male relatives and to compare its potential with the commonly used Yfiler set, we first calculated the observed mutation rate for each of the two Y-STR sets using a Bayesian approach. For each set of Y-STRs, the number of mutations observed in each father-son pair was modeled using a Poisson distribution. For the RM Y-STRs, the median mutation rate of the posterior distribution was estimated to be 1.97x10 -2 (95% CI 1 .8x10 -2 - 2.2x10 -2 ), which is the estimated median rate of Yfiler Y-STR (3.0x10 -3 (Range is 2.39x10 -3 Up to 3.72x10 -3 Next, for a given father-son pair, the probability of observing at least one mutation in each of the two Y-STR sets was estimated, reflecting the minimum standard for distinguishing male relatives. Assuming that all Y-STRs in each set were successfully genotyped and using a posterior estimate of the mutation rate for each set of Y-STR markers, the probability of observing at least one mutation in the RM Y-STR set was 0.1952 (95% CI of 0.177 to 0.21). Unexpectedly, this value is more than 4 times the value estimated for the Yfiler set, which is 0.047 (95% CI of 0.038 to 0.057). The probability of observing at least one mutation in the RM Y-STR set was statistically significantly higher than the probability in the Yfiler set (p<5.0x10 -07). Finally, samples were experimentally tested, independently of those used to establish mutation rates, whether the new RM Y-STR panel could be used in practice to distinguish male relatives. To this end, 103 male relative pairs from 80 male pedigrees (related between 1 and 20 generations within their pedigrees) were genotyped and the results compared with those obtained from the Yfiler kit on the same samples. Overall, the RM Y-STR panel distinguished 70.9% of male relative pairs by at least 1 mutation, reflecting a level of male relative discrimination that was 5 times higher than that of the Yfiler kit panel (only 13%); notably, the significant difference (t = 6.389, p < 0.0001) was similar to the statistical expectations for the initial father-son pair analysis ( Figure 4 and Table 3). Within pedigrees, the RM Y-STR set distinguished 70% of father-son pairs, 56% of brothers, and 67% of cousins ​​( Figure 4 and Table 3). In contrast, the Yfiler group was unable to distinguish any father-son pairs or cousins ​​in the data set and was only able to distinguish 6% of the brothers ( Figure 4 and Table 3). Furthermore, all relatives separated by more than 11 generations could be distinguished by 1 or more mutations using the RM Y-STR panel, but only 33% using the Yfiler panel.

[0067] All compositions and methods disclosed and claimed herein can be prepared and implemented according to the present disclosure without excessive experimentation. Although the compositions and methods of the present invention may be described by specific examples or preferred embodiments, these examples and embodiments are by no means intended to limit the scope of the claims in any way, and it will be clear to those skilled in the art that the compositions and / or methods described herein and the steps or the order of steps of the methods can be changed without violating the idea, spirit and scope of the present invention. More specifically, it will be clear that certain chemical and physiologically related reagents can replace the reagents described herein, and the same or similar results will be obtained at the same time. All such similar substitutions and changes that are clear and obvious to those skilled in the art will be considered to be within the spirit, scope and idea of ​​the present invention defined by the appended claims.

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Claims

1. Use of an amplification primer pair set in preparing a kit for distinguishing a father-son pair, the amplification primer pair set comprising primers for amplifying a Y-STR marker, wherein the Y-STR marker consists of: DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627.

2. A kit for distinguishing father-son pairs by identifying the alleles of Y-chromosome STR markers, the markers consisting of: DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627, and the kit includes primers for amplifying all loci, as well as an allelic ladder representing the markers.

3. Use of an allele ladder size standard in the preparation of a kit for distinguishing father-son pairs, the standard comprising a size standard of one or more alleles of a Y-STR marker, the Y-STR marker consisting of: DYF387S1, DYF399S1, DYF403S1, DYF404S1, DYS449, DYS518, DYS526, DYS547, DYS570, DYS576, DYS612, DYS626, and DYS627.

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