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DIP – STR : Highly Sensitive Markers for the Analysis of Unbalanced Genomic Mixtures

2013; Wiley; Volume: 34; Issue: 4 Linguagem: Inglês

10.1002/humu.22280

1098-1004

Vincent Castella, Joëlle Gervaix, Diana Hall,

Genomic variations and chromosomal abnormalities

Human MutationVolume 34, Issue 4 p. 644-654 MethodsOpen Access DIP–STR: Highly Sensitive Markers for the Analysis of Unbalanced Genomic Mixtures Vincent Castella, Vincent Castella Unité de Génétique Forensique, Centre Universitaire Romand de Médecine Légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, 1011 Switzerland These authors contributed equally to this work.Search for more papers by this authorJoëlle Gervaix, Joëlle Gervaix Unité de Génétique Forensique, Centre Universitaire Romand de Médecine Légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, 1011 Switzerland These authors contributed equally to this work.Search for more papers by this authorDiana Hall, Corresponding Author Diana Hall Unité de Génétique Forensique, Centre Universitaire Romand de Médecine Légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, 1011 SwitzerlandCorrespondence to: Diana Hall, Unité de Génétique Forensique, Centre Universitaire Romand de Médecine Légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Rue du Bugnon 21, Lausanne 1011, Switzerland. E-mail: Diana.Hall@chuv.chSearch for more papers by this author Vincent Castella, Vincent Castella Unité de Génétique Forensique, Centre Universitaire Romand de Médecine Légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, 1011 Switzerland These authors contributed equally to this work.Search for more papers by this authorJoëlle Gervaix, Joëlle Gervaix Unité de Génétique Forensique, Centre Universitaire Romand de Médecine Légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, 1011 Switzerland These authors contributed equally to this work.Search for more papers by this authorDiana Hall, Corresponding Author Diana Hall Unité de Génétique Forensique, Centre Universitaire Romand de Médecine Légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Lausanne, 1011 SwitzerlandCorrespondence to: Diana Hall, Unité de Génétique Forensique, Centre Universitaire Romand de Médecine Légale, Centre Hospitalier Universitaire Vaudois et Université de Lausanne, Rue du Bugnon 21, Lausanne 1011, Switzerland. E-mail: Diana.Hall@chuv.chSearch for more papers by this author First published: 25 January 2013 https://doi.org/10.1002/humu.22280Citations: 35 Communicated by Pui-Yan Kwok AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat ABSTRACT Samples containing highly unbalanced DNA mixtures from two individuals commonly occur both in forensic mixed stains and in peripheral blood DNA microchimerism induced by pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred as short tandem repeats (STR), and single-nucleotide polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP–STR that relies on pairing deletion–insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP–STR genotypes of the minor were successfully procured at ratios up to 1:1,000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP–STR approach on an initial set of nine markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures. Introduction Genetic polymorphisms such as short tandem repeats (STR) and single-nucleotide polymorphism (SNP) are commonly used in forensic identity testing [Gill et al., 1994, 2001; Jobling and Gill, 2004; Kayser et al., 1997; Moretti et al., 2001; Tully et al., 2001] and medical genetics [Goate et al., 1991; Hastbacka et al., 1992; Houwen et al., 1994; Wooster et al., 1995]. However, because of PCR-based analytical methods, these markers have low sensitivity in characterizing samples that contain DNA from several contributors in very different proportions. In general, PCR fragment analysis that is not deep sequenced allows for the detection of a minor DNA component in a mixture only when it represents more than 10% of the total DNA; however, the unambiguous identification of all minor DNA alleles requires a minor DNA fraction of at least 20% [Fregeau et al., 2003; Sutherland et al., 2009; Westen et al., 2009]. Historically, characterizing unbalanced DNA mixtures has been associated with the analysis of biological stain for forensic identification purposes. For example, challenging mixed stains may be derived from samples including, but not limited to, clothing, hair, skin, or items the perpetrator may have touched. Because these samples are likely to carry small quantities of the perpetrator's DNA mixed with a large amount of the victim's DNA, the limit of resolution of currently used markers may have dramatic consequences for justice. Moreover, several fields of medical genetics recently expressed a paramount need for tools that enable the analysis of unbalanced DNA mixture occurring in vivo, also referred as DNA microchimerism (less than 1% of foreign cells). Few examples are the DNA microchimerism associated to pregnancy, which is caused by the transient circulation of minute quantities of fetal DNA in the maternal blood [Lo et al., 1998; Tjoa et al., 2006] or the trace quantities of donor's DNA in the body fluids (blood and urine) of transplanted patients [Chok et al., 2002; Gadi et al., 2006; Moreira et al., 2009; Pujal and Gallardo, 2008]. Advances in the above medical fields all require novel analytical approaches for the detection and quantification (specific for organ transplant applications) of the DNA of interest when mixed to a high foreign DNA background. A simple solution to this problem is represented by a standard amplification-based method that targets a genomic region unique to the minor DNA (allogeneic marker) eliminating the masking effect of the major DNA. Molecular markers located on the Y chromosome are the most widely employed allogeneic markers for all cases of male DNA detection over high female background [Roewer, 2009]. Unfortunately, this approach has several limitations: first, the applicability to mixtures with a specific sex mismatch dramatically reduces the number of suitable cases. Second, because of the mostly nonrecombining nature of the Y chromosome, without mutations paternally related individual all share the same Y STR alleles. Third, since all Y STR constitute a single haplotype, multiplying the individual allele frequencies is not valid as for independently inherited autosomal STR. Therefore, a match between Y STR profiles that is evaluated on the basis of haplotype frequencies may be an evidence of reduced weight for forensic identification purposes [Vermeulen et al., 2009]. On this issue, a sequencing-based study of two Y chromosomes separated by 13 generations discovered four single-base differences in 10 Mb DNA [Xue and Tyler-Smith, 2010]. This suggests that the Y chromosome accumulates around one mutation per generation, which means that a sequencing-based assay should distinguish almost every Y chromosome. Alternatively, it was recently proposed a locus-specific approach, which is based on the analysis of 13 rapidly mutating Y-STRs. Although this method can be more easily executed, the results indicate a lower performance with about 50% of father and sons being distinguished among 305 male relatives [Ballantyne et al., 2012]. The human leukocyte antigen (HLA) gene variants are another example of allogeneic markers, and are typically employed in transplantation follow-up studies [Gadi et al., 2006]. Nevertheless, this method uses a genetic marker that has an effect on the immunogenic compatibility of the recipient–donor pair; therefore, it necessarily introduces an ascertainment bias in the microchimerism analysis induced by organ transplant. Additionally, as for the Y chromosome, HLA variants are clustered on the same chromosome, resulting in a decreased power of discrimination between individuals. In peripheral blood DNA macrochimerism (usually more than 10% of foreign cells) induced by hematopoietic stem cell transplantation, new assays have been proposed for all donor–recipient type based on biallelic deletion–insertion polymorphisms (DIP) polymorphisms and null alleles [Alizadeh et al., 2002; Jimenez-Velasco et al., 2005]. Yet, biallelic systems are associated with a low discrimination power which makes them sensitive to confounding factors such as transfusions associated to the surgery, inborn microchimerism [Rubocki et al., 2001] or other possible medically related contaminations linked to the amplification of minute quantities of DNA. Finally, great expectations are placed on the contribution of next-generation sequencing techniques to forensic and DNA microchimerism analyses [Irwin et al., 2011; Snyder et al., 2011]. However, a recent publication indicates that caution must be taken [Bandelt and Salas, 2012]. These authors conducted an indirect quality assessment of a study on heteroplasmic mitochondrial DNA mutations based on high-throughput sequencing [He et al., 2010]. By using in silico phylogenetic approaches, they found that on average at least five mutations were missed per sample. Although this is one particular study and the error rate may dramatically reduce with the rigorous application of quality controls and standards to be defined, next-generation sequencing may remain of limited use for immediate forensic applications. Cautions are mainly about: the quantity and quality of DNA required; the persistence of PCR bias as forensic applications is expected to use target sequencing approaches (not whole genome); the risk of cross contamination is due to a large number of parallel reactions; the difficulty of repeat sequence analysis (STR) and finally; the times, costs, and expertise required for this type of analysis [Berglund et al., 2011; Hert et al., 2008; Metzker, 2010; Snyder et al., 2011]. With these considerations in mind, we propose here new genetic markers, the DIP–STR, which are located throughout the genome, highly polymorphic, easy to genotype, and capable of resolving extremely unbalanced two DNA mixtures (ratio 1:1,000). We describe an initial set of DIP–STR markers including a population survey and data of their performance on both simulated DNA mixtures and two representative clinical samples. Further, we discuss the potential contribution of this tool to various research fields. Methods Database Search of DIP-Linked STR Markers From the University of California Santa Cruz (UCSC) database ([assembly Feb2009(GRCh37/hg19]) [Kent et al., 2002], by using the "Table Browser" tool, we searched for DIP polymorphisms in the group "Variation and Repeats," track "SNPs (131)", region "genome" filtered by class "in-del", "insertion" and "deletion"; those with "unknown" validity were excluded and we set sequence "weight" equal to 1. About 216,000 DIP were obtained on the basis of these criteria, this number still included stretches of the same base and short nucleotide repeats, which we excluded after selection of DIP-linked STR. From the same database, STR were selected from the group "Mapping and Sequencing Tracks", track "STS Markers" filtered for the presence on the "Marshfield genetic map" by selecting those with assigned "MarshfiledChrom" and "MarshfieldPos", we further eliminated those with a Marshfield chromosome assignment not corresponding to the UCSC chromosome assignment. About 7,469 STR were selected on the basis of these criteria. The DIP characterized by a deletion/insertion of at least 2 bp located less than 500 bp from an STR were about 70. To ensure the independence of markers and to reduce the risk of linkage with disease or other phenotypic information, we searched for candidate DIP–STR located on different chromosomes (when possible) and in noncoding regions, which are not clinically associated. Once an initial list of nine DIP–STR markers was established (Table 1), we tested single-marker allelic variability by genotyping 20 Swiss individuals under informed consent. Table 1. DIP–STR Marker List DIP–STR Chromosome DIP S/L sequence STR repeat DIP–STR size (bp) MID1013aa Marker name from the Marshfield database correspond to rs1611095, rs2308142, and rs2067195, respectively.–D5S490 5q23.2 –/CCAG GT 307–343 MID1950aa Marker name from the Marshfield database correspond to rs1611095, rs2308142, and rs2067195, respectively.–D20S473 20p13 –/ATT TTA 213–231 MID1107aa Marker name from the Marshfield database correspond to rs1611095, rs2308142, and rs2067195, respectively.–D5S1980 5p15.33 –/AACA CA 650–680 rs11277790–D10S530 10q25.1 –/TCCAACT GT 344–362 rs60194384–D15S1514 15q26.2 –/TCTTAA TATC 281–309 rs67842608–D5S468 5q11.2 –/TGGTTTAA GT 379–395 rs66679498–D2S342 2q32.3 –/CCAACTTTCTCCTAC CA 340–357 rs10564579–D3S1282 3p24.1 –/GTCATA CA 714–728 rs35708668–D5S2045 5q34 –/TACTATGTAC CA 621–649 a Marker name from the Marshfield database correspond to rs1611095, rs2308142, and rs2067195, respectively. Primer Design and PCR Conditions Each single DIP and STR polymorphism was first independently validated by PCR. To do so, we used the primer3 software [Rozen and Skaletsky, 2000] for designing primers around the DIP polymorphisms; we used the primers indicated on the UCSC database for the STR. The L-DIP primers were designed to include a 3′end region that was complementary to the inserted sequence, whereas the S-DIP primer lacked the inserted sequence at the 3′end and passed the insertion point by three to seven nucleotides (Supp. Table S1). It should be noted that a condition for strong specificity of both S and L primers is that the inserted sequence and the region past the insertion point are substantially different. Each PCR reaction was performed in a final volume of 20 µl containing 1× PCR buffer with 1.5 mM MgCl2 (Applied Biosystems, Zug, Switzerland), 250 µM of each dNTP, 1.2 U AmpliTaq Gold DNA Polymerase, 1 µM of each forward and reverse primer and 1 ng of genomic DNA (DNA quantity varied for specificity and limit of detection tests). The PCR thermocycling conditions are: 5 min at 95°C; followed by 60 sec at 94°C, 60 sec at 52°C, and 60 sec at 72°C for 30 cycles and a final extension of 30 min at 72°C. The annealing temperature of 52°C was modified to 58°C when using DIP–STR primers MID1107-D5S1980 and both annealing and extension time were 75 and 90 sec for marker MID1107-D5S1980 and rs10564579-D3S1282 and rs35708668-D5S2045. The thermal cyclers employed are GeneAmp 9700 (Applied Biosystems, Zug, Switzerland). Before capillary electrophoresis, 1 µl PCR product is added to 8.5 µl deionized formamide HI-DI (Applied Biosystems, Zug, Switzerland) and 0.5 µl GS-ROX 500 size standard (Applied Biosystems, Zug, Switzerland). DNA fragments are separated using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Zug, Switzerland) according to manufacturer's instruction and analyzed with GeneMapper ID v3.2 software (Applied Biosystems, Zug, Switzerland), with minimum interpretation peak threshold of 50 relative fluorescence units (RFU). Allele Specificity Tests Amplification specificity, when using S-DIP and STR primers as well as L-DIP and STR primers, was tested by amplifying 1 ng of DNA template heterozygous for the DIP allele mixed with increasing quantities of a second DNA homozygous for the opposite DIP allele LL and SS respectively (as "informative genotype 2" in Fig. 1A), both DNA were selected irrespective of the genotype of the linked STR. The DNA ratios tested included 1:10, 1:20, 1:50, 1:100, and 1:1,000. One representative result is reported in Figure 2 together with the comparative analysis of the same mixture amplified by a standard forensic kit, NGMselect (Applied Biosystems, Zug, Switzerland) according to manufacturer's instruction. Figure 1Open in figure viewerPowerPoint A: DIP–STR informative genotypes. A DIP–STR haplotype is analyzed by using PCR primers overlapping the DIP on one side (either L-DIP or S-DIP primer) and downstream the STR (STR primer) on the other side. When major and minor DNA contributors are opposite homozygous LL/SS or SS/LL ("informative genotype 1"), two minor DNA haplotypes can be identified in the mixture (red box); conversely, when the major DNA contributor is homozygous, either SS or LL, and the minor DNA contributor is heterozygous ("informative genotype 2"), one minor DNA haplotype can be identified in the mixture (blue box). Arrows indicate PCR primers. B: Theoretical evaluation of the occurrence of informative markers. Letter s and l indicate the allele frequencies of S and L alleles, respectively. In red are the probabilities of informative DIP genotypes enabling the identification of two minor DNA haplotypes ("informative genotype 1"). Depending on the linked STR alleles, these are the same or different. In blue are the probabilities of informative DIP genotypes enabling the identification of one minor DNA haplotype ("informative genotype 2"). The sum of these values (s2l2+l2s2+2s3l+2sl3) gives the probability of having any type of informative genotype (I) in a mixture of two DNA. Figure 2Open in figure viewerPowerPoint Performance of DIP–STR and forensic STR in resolving various ratios of DNA mixtures. A: The electropherograms show the DIP–STR-specific amplification of a minor DNA when mixed to increasing quantities (1:10 to 1:1,000) of a second DNA, both genotypes of the two-mixed DNA are known. We report here one informative marker, rs67842608-D5S468 as a representative example. The minor DNA-specific amplification is achieved by using the S-DIP primer, which targets the minor allele 92S (377 bp), that is not shared with the major DNA, 90L 94L (383–387 bp). Note that the alleles of the major DNA are 375 and 379 bp long, when they are amplified by the S-DIP primer due to a nonspecific amplification. B: The same DNA mixtures were analyzed by the forensic kit NGMselect (Applied Biosystems). We show here only four representative markers. The electropherogram corresponding to the DNA ratio 1:10 shows that the minor DNA alleles indicated by red arrows are close to the detection limit and they are quickly not detected anymore when the quantity of the major DNA increase to 1:20, 1:50, and so on (for 1:100 and 1:1,000 data are not reported). PCR Limit of Detection We estimated the minimal amount of DIP heterozygous DNA required to produce a DIP–STR product detectable by capillary electrophoresis, by varying the total DNA content in the PCR reaction from 1 down to 0.025 ng. Amplifications were done in duplicate, and the number of PCR cycles was increased to 34. Allele Frequencies and Summary Statistics We genotyped 103 Swiss unrelated individuals with the nine DIP–STR markers of Table 1. Blood and saliva samples were taken after informed consent and approval from the local ethic committee. DNA was extracted by using the QIAamp DNA Mini kit (Qiagen AG, Basel, Switzerland) according to the manufacturer's guidelines and quantified using the Quantifiler Human DNA Quantification Kit (Applied Biosystems, Zug, Switzerland). Summary statistics of population survey is reported in Table 2. Haplotype frequencies are indicated in Supp. Table S2. CEPH 1347–02 DNA was genotyped as a reference control for allele size. Its genotype is indicated at the bottom of each marker frequency data. The marker haplotypes are named according to the S or L allele at the DIP polymorphism and the allele size of the linked STR marker when this is analyzed by STR primers of Supp. Table S1. For markers MID1013-D5S490, MID1950-D20S473, and MID1107-D5S1980, the STR allele name is expressed in arbitrary numbers increasing according to the numbers of STR repeats. Table 2. DIP–STR Marker Diversity DIP–STR Haplotype, N S haplotype, N (frequency) L haplotype, N (frequency) Obs. Het. I MID1013–D5S490 15 6 (0.78) 9 (0.22) 0.49 0.28 MID1950–D20S473 10 5 (0.62) 5 (0.38) 0.80 0.36 MID1107–D5S1980 13 8 (0.31) 5 (0.69) 0.74 0.34 rs11277790–D10S530 15 8 (0.81) 7 (0.19) 0.73 0.26 rs60194384–D15S1514 12 7 (0.61) 5 (0.39) 0.88 0.36 rs67842608–D5S468 13 4 (0.21) 9 (0.79) 0.62 0.28 rs66679498–D2S342 11 6 (0.65) 5 (0.35) 0.69 0.35 rs10564579–D3S1282 12 7 (0.73) 5 (0.27) 0.85 0.32 rs35708668–D5S2045 19 18 (0.96) 1 (0.04) 0.85 0.07 N, number; Obs. Het., observed heterozygosity; I, probability of informative genotypes. Clinical Samples Blood and saliva samples were collected under informed consent and approval from the local ethics committee, which required for the forensic sample the additional agreement of the justice office responsible of the investigation. Forensic mixed stain collected on the body of the victim and reference samples from the victim (blood) and the three suspects (saliva) were extracted with the QIAamp DNA mini kit following manufacture's instruction. PCR genotyping and electrophoresis were performed as indicated in the paragraph "Primer design and PCR conditions." We used 4 ng of total DNA from the mixed stain to amplify DIP–STR markers. Pregnancy DNA microchimerism samples were obtained from 2 ml of maternal plasma extracted by QIAamp DNA mini kit according to manufacture's instruction. DNA was eluted in 60 µl of H2O, 6 µl of DNA was used for PCR. Reference samples of the mother (blood) and the father of the baby (saliva) were extracted by QIAamp DNA mini kit following manufacture's instruction. Forensic autosomal and Y chromosome STR were analyzed by using the AmpFlSTR SGM Plus PCR Amplification Kit (Applied Biosystems, Zug, Switzerland) and the Powerplex Y Amplification kit (Promega, Dubendorf, Switzerland) according to manufacturer's instruction. Analysis of DIP–STR Markers' Performance To evaluate the discrimination power of DIP–STR markers, we used real DIP–STR genotypes observed in 103 individuals to simulate in silico 5,253 pairwise DNA mixtures. For each pair of DNA, we considered both possibilities of major and minor DNA contributors for a total of 10,506 simulated DNA mixtures. Marker rs35708668-D5S2045 was eliminated because of the low information content (L haplotype frequency of 0.04). Based on these data, we counted the number of markers showing informative genotypes. The DIP–STR haplotypes of a minor DNA contributor that are nonshared with the major DNA represents the DNA profile that can be detected in the mixture. In Table 3 column 1, we summarized these results by indicating the percentage of simulated DNA mixtures showing a given minimum number of informative markers. Table 3. Occurrence of Informative Markers Empirical estimate using eight DIP–STRaa Marker rs35708668-D5S2045 was eliminated because of the extremely low information content. Expected estimate using 30 DIP–STR Percentage of DNA mixtures (≥N informative markers) 95 (≥1) 95 (≥6) 76 (≥2) 89 (≥7) 47 (≥3) 79 (≥8) 21 (≥4) 66 (≥9) 6 (≥5) 50 (≥10) a Marker rs35708668-D5S2045 was eliminated because of the extremely low information content. Alternatively, a theoretical estimate of the DIP–STR informativeness is also possible given the fact that the occurrence of informative genotypes depends on the presence of DIP alleles, which are unique to the minor DNA contributor. Based on Hardy–Weinberg assumptions, the probability of informative genotypes (I) at a given DIP–STR marker can be calculated as I = 2s2l2+2s3l+2sl3 (Fig. 1B). Where s and l are the frequencies of the S and L alleles, 2s2l2 is the probability that major and minor DNA contributors are homozygous for the opposite DIP allele (s2l2 +l2s2); whereas, 2s3l+2sl3 are the probabilities that the major DNA contributor is DIP homozygous either S or L and the minor DNA contributor is DIP heterozygous, respectively ([s2[2sl]+ l2[2sl]). The I value for the current nine DIP–STR markers is reported in Table 2. As average, our markers show a probability of being informative of 0.32, after the exclusion of the least polymorphic marker (rs35708668-D5S2045). In Table 3 column 2, we calculated the theoretical percentage of DNA mixtures with at least six to 10 informative markers by using a DIP–STR panel of 30 loci. This is based on the cumulative binomial distribution of 30 trials (markers) each one associated to a probability of being informative (success of the trial) of 0.32. Finally, we calculated the corresponding match probability of this initial set of markers. For each informative marker of a simulated DNA mixture (see above), we considered the frequency of the matching genotypes in the population. When a marker is informative of two minor alleles (Fig. 1A, "informative genotype 1"), this corresponds to the frequency of the heterozygous individual in the population. Conversely, when the marker is informative of only one minor DNA allele (Fig. 1A, "informative genotype 2"), the frequency of the matching genotype includes the frequency of the corresponding homozygous individual in addition to all of the possible heterozygous individuals who carry the observed allele together with any of the DIP opposite haplotypes. Fore each DNA mixture, the results of informative marker were multiplied under the assumption of independence of DIP–STR haplotypes and unrelated individuals. In the results section, we report a summary of the matching probability distribution calculated across 10,506 DNA mixtures. Results Principle of the Method To circumvent the problem of the major DNA contributor masking the minor DNA of a mixture, we propose here a PCR-based method characterized by allele-specific primers capable of targeting DNA sequences, which are unique to the minor DNA. These sequences are biallelic DIP of few nucleotides, mostly between three and 15. The two possible alleles are also referred as, long allele (L) and short allele (S). Because biallelic markers have reduced information content, we propose the selection of DIP linked to STR to form a compound marker termed DIP–STR. The multiallelic haplotype composed of both DIP and STR alleles is analyzed by using PCR primers overlapping the deleted–inserted sequence on one side and downstream the STR region on the other side (Fig. 1). Studies of patterns of human linkage disequilibrium and recombination hotspots indicate that a distance of few hundred base pairs ensures the absence of recombination between DIP and STR [Gabriel et al., 2002; Reich et al., 2001]. The novelty of this marker with respect to other existing compound genetic markers (SNP–STR for example) is the use of extended sequence polymorphisms (3–15 bp of the DIP), which allow the sensitive and specific amplification of the minor DNA contributor in the presence of large quantities of foreign DNA background. For a given DNA mixture, a DIP–STR marker can be informative or uninformative depending on the S/L allele mismatch between the two DNA to be discriminated (Fig. 1A). When major and minor DNA are opposite homozygous for the DIP allele, both DIP–STR haplotypes of the minor DNA can be targeted by allele-specific PCR. These minor DNA haplotypes may be identical or different depending on the homozygosity or heterozygosity of the linked STR (Fig. 1A, "informative genotype 1"). Instead, when major and minor DNA are homozygous and heterozygous, respectively, for the DIP alleles, the DIP–STR marker is still informative but the number of minor DNA haplotype that can be identified is only one (Fig. 1A, "informative genotype 2"). Conversely, uninformative cases arise when the minor DNA has no unique S or L allele. This occurs when major and minor DNA are homozygous for the same DIP allele or each time the major DNA is heterozygous SL. DIP–STR List and Haplotype Frequencies We developed here nine DIP–STR markers characterized by DIP to STR distance ranging between 213 and 728 bp (Table 1). The deleted–inserted sequences range between 3 and 15 bp. Seven STR are di-nucleotide repeats, one tri-, and one tetra-nucleotide repeats. Six selected markers span different chromosomes and three are located on distant regions of chromosome 5 (5q11.2, 5q23.2, and 5q34). In Table 2, we report values of marker variability obtained from a survey of 103 unrelated Swiss individuals. The alleles of this compound marker correspond to the observed haplotypes formed by DIP and STR alleles. The observed heterozygosity range from 0.49 to 0.88. For the nine DIP–STR of Table 2, the number (N) of observed haplotypes varies between 10 and 19 and the frequencies of S versus L containing haplotypes are comparable except for marker rs35708668-D5S2045, where a single L haplotype was observed in the population surveyed. PCR Limit of Detection and Specificity Tests To examine the minimal amount of DNA template required for successful amplification using S-DIP and STR primers as well as L-DIP and STR primers, we amplified serial dilutions of DNA template from individuals heteroz