Multiplex PCR development of Y-chromosomal biallelic polymorphisms for forensic application

Single-nucleotide polymorphisms of Y chromosome (Y-SNPs) are a class of markers of interest in forensic investigations, because many of them show regional specificity, providing useful information about the geographic origin of a subject or evidence under investigation. A first multiplex with 7 SNPs (M35, M89, M9, M170, M172, M45, M173), which occur in the basal branches of the phylogenetic tree and are able to assign a subject to known most frequent European haplogroups, was designed. SNP genotyping was accomplished by hot-start PCR with primers amplifying fragments between 96 and 136 nucleotides, minisequencing, and capillary electrophoresis of extension products. Ninety seven subjects of known geographic provenance were studied, of which 68 from Europe. Of these, 57 had mutations found more frequently in European haplogroups and 11 more frequent in Asian populations. Subjects from non-European countries were also examined and had haplogroups common in their regions of provenance. Experiments with low molecular weight DNA gave positive amplification from 1 ng of DNA for all seven SNPs.     

Y-SNPs analysis using two different approaches in a Northern Portugal male population sample

The non-recombining portion of the human Y (NRY) chromosome has various types of variation, including single nucleotide polymorphism (SNP). In spite of their low discrimination power, they provide a powerful and simple exclusion tool for forensic purposes. A special advantage of SNPs is that it can potentially detect smaller DNA fragments (analysis of degraded DNA).The aim of this work consisted in the analysis of a group of SNP polymorphisms (M2, M9, M35, M89, M45, M170, M172, M173, M207 and P25) in a Northern Portugal male population sample, which allows the determination of the most common European haplogroups, including the Northern Portugal ones.The method used for typing these polymorphisms was the real-time PCR with TaqMan probes on the ABI 7000 platform (Applied Biosystems).We had some difficulties in typing some of the markers using this approach. However, the preliminary results obtained for the defined haplogroups are in accordance with those described in close European populations. To confirm the typing and solve the doubts that emerged from the real-time approach, the samples were also typed using SNapShot.     

武汉汉族8个Y染色体双等位基因标记遗传多态性

目的筛选汉族群体中具有多态性的Y染色体双等位基因标记并研究其等位基因及单体群频率分布, 为法医学应用和群体进化研究提供基础数据。方法采用片段长度差异等位基因特异性PCR和PAGE技术对武汉地区160名男性汉族无关个体的8个Y染色体双等位基因标记(M9、M89、M111、M119、M122、M134、IMS-JST003305 和SRY+465)进行分型。结果 8个双等位基因标记在武汉汉族群体中均具有遗传多态性,其基因多样性(GD)范围为 0．0126-0．4830,共检出9种不同单体群(Hg1～9),其单体群多样性(HD)为0．7776。结论 8个Y染色体双等位基因标记组成的单体群在法医学应用和群体进化研究中具有一定的实用价值,可作为Y-STRs标记的有效补充。     

EA-YPredictor:基于Y-STR数据的家系特异性单倍群归属判别分析软件

目的Y染色体为男性所特有,其遗传标记蕴含着丰富的生物地理信息,故可溯源家系,在嫌疑人排查和追踪中发挥作用。Y-STR突变率较高,而Y-SNP突变率极低,几乎不会发生回复突变,所以后代男性群体携带祖先特有的Y-SNP。本研究期望通过现在我国Y库建设中通用的17个Y-STR的单倍型数据预测Y-SNP单倍群细支。方法基于前期观察,选取千人基因组计划III期中的513例东亚人群(中国及周边区域)作为基础数据集,在Java平台和Microsoft Excel软件框架下,以遗传距离计算和Y染色体进化树构建手段相联合研发Y-STR数据的家系特异性单倍群归属判别分析软件:EA-YPredictor。结果本研究揭示了15个单倍群大支下的核心单倍型。通过随机选取70个公开数据库样本,EA-YPredictor软件预测准确性达到92.8%(95%置信区间:[84.1%,97.6%])。结论在Y-SNP复合扩增检测尚无定论的情况下,本软件可基于二代测序样本对Y-STR数据库样本进行单倍群细支的准确预测,能适用于辅助家系单倍群判断。随着测序技术的不断换代和优化,更多高通量的Y-STR和Y-SNP数据补充将会使本软件进一步优化。此外,本软件对于Y数据库中Y-SNP遗传标记的筛查建库有一定指向作用。     

Y-SNP typing of U.S. African American and Caucasian samples using allele-specific hybridization and primer extension

Multiplex analysis of genetic markers has become increasingly important in a number of fields, including DNA diagnostics and human identity testing. Two methods for examination of single nucleotide polymorphisms (SNPs) with a potential for a high degree of multiplex analysis of markers are primer extension with fluorescence detection, and allele-specific hybridization using flow cytometry. In this paper, we examined 50 different SNPs on the Y-chromosome using three primer extension multiplexes and five hybridization multiplex assays. For certain loci, the allele-specific hybridization method exhibited sizable background signal from the absent alternate allele. However, 100% concordance (>2000 alleles) was observed in ten markers that were typed using both methods. A total of 18 unique haplogroups out of a possible 45 were observed in a group of 229 U.S. African American and Caucasian males with the majority of samples being assigned into 2 of the 18 haplogroups.     

47个SNPs复合检测方法的建立及其法医学应用

目的建立47-plexSNPs复合检测方法，评价其在法医学中的应用价值。方法筛选46个常染色体SNPs和1个Y—SNPs，使用2个检测体系分别对47个SNPs进行单管内复合PCR扩增，采用荧光标记单碱基延伸法和毛细管电泳检测技术进行分型检测；并用建立的方法对260份广东地区无关个体血样进行47个SNPs分型。结果建立的47-plex SNPs的复合检测体系灵敏度高，种属特异性好；260名个体所有SNPs均能准确分型，群体内基因型频率分布均符合Hardy—Weinberg平衡，累积个人识别率大于0．9999，累积非父排除率为0．99982，累积偶合率为6．24×10一。结论本文47-plex SNPs复合检测方法能同时对47个SNPs进行快速、准确的检测，在法医学个体识别鉴定中具有良好的应用前景。     

Targeted Y chromosome capture enrichment in admixed South American samples with haplogroup Q

Y haplogroups, defined by Y-SNPs, allow the reconstruction of the human Y chromosome genealogy. Recently, MPS based panels were introduced in the forensic genetics community for Y-SNP typing and identification of a broad range of haplogroups. The panels are based on an amplicon strategy and allow the detection of up to 15,600 Y-SNPs. The panels target up to 210,000 bps, which should be compared to the overall 8.9 Mbps comprising the unique regions of the non-recombining portion of the Y chromosome (NRY). We present an alternative approach of sequencing unique regions within the NRY using target enrichment probes and hybridization capture. A total of 359,954 probes were designed using the SureDesign software, representing 7.5 Mbps of the NRY. Library preparation and capture were performed using the Agilent SureSelect XT HS2 Target Enrichment method and sequencing was performed in a NovaSeq 6000 System. Besides individual barcodes, the method also included unique molecular barcodes for additional quality screening. The method was tested on admixed South Americans that carry a Y chromosome of haplogroup Q. We successfully identified novel variation that could potentially help refining haplogroup Q phylogeny.     

Grouping of Y-STR haplotypes discloses European geographic clines

Y-STR haplotypes are widely studied in Europe and an extensive databasing effort has been conducted (http://www.ystr.org). The distribution of these haplotypes has been considered to present no evidence for substructure at central and southern European level. This picture contrasts with the one that results from Y haplogroups defined by binary markers. This paradox has been solved by admitting that the high STR mutation rate and corresponding recurrence has erased geographic structuration. This explanation prompted us to reanalyse Y-STR haplotypes distribution bearing in mind the commonly admitted model for the generation of diversity in these markers, namely the stepwise mutation model (SMM) and, thus, taking the molecular distance between haplotypes into consideration. Accordingly, we have studied the European distribution of the two most frequent haplotypes in the Iberian Peninsula and their one step neighbours using the European samples deposited in the Y STR database (http://www.ystr.org). For the first group we found a clear-cut decreasing W-E gradient, while for the second the highest frequencies were found in the Iberian Peninsula (3.98% in Portugal and 3.85% in Spain), dropping to 2.88% in France and showing a less well defined SW-NW gradient. Furthermore, we have tested the agreement between haplotype groups and binary markers haplogroups in a random sample of 292 individuals from Northern Portugal. Our results demonstrate that (a) Y-STR haplotype data can be used for wide-scale anthropological approaches disclosing information that has been considered only available through binary markers and (b) forensic use of continental databases needs careful refinement, due to the macro-geographic pattern now evidenced.     

Routine Y-STR typing in forensic casework

In the field of molecular diagnosis, forensic casework analysis is one of the most demanding investigations, due to its social impact. Optimization of DNA typing multiplex reactions with identical cycling conditions as those required by autosomal short tandem repeats (STR) multiplex reduces errors, and saves time and reagents. Previously, we validated a five Y-STRs set, all of them generating single band patterns. This work reports the optimization of combined multiplexes, a triplex (DYS19, DYS390 and DYS391) and a duplex (DYS392 and DYS393), that can be amplified in identical cycling conditions as those required by commercially available multiplex autosomal STR kits. In addition both Y chromosome multiplexes can be combined for co-injection on a capillary electrophoresis based automated sequencer. Statistical attributes of the haplotypes of the five Y-STR investigated were evaluated in unrelated males from different metropolitan areas of Argentina. This system was successfully used for investigating more than 350 forensic routine cases in our country.     

A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers

A multiplex polymerase chain reaction (PCR) assay capable of simultaneously amplifying 20 Y chromosome short tandem repeat (STR) markers has been developed to aid human identity testing and male population studies. These markers include all of the Y STRs that make up the "extended haplotype" used in Europe (DYS19, DYS385, DYS389I/II, DYS390, DYS391, DYS392, DYS393, and YCAII) plus additional polymorphic Y STRs (DYS437, DYS438, DYS439, DYS447, DYS448, DYS388, DYS426, GATA A7.1, and GATA H4). Primers for the markers DYS385, DYS389, and YCAII target duplicated regions of the Y chromosome and thus can provide two polymorphic peaks for each respective primer set. This Y STR 20plex, which utilizes 34 different PCR primers, is the first to include a simultaneous amplification of all the markers within the European "minimal" and "extended" haplotypes. Relative primer positions are compared between the newly developed primers described here and previously published ones. Efforts were made to avoid X chromosome homology in the primer design as well as close packing of PCR product size ranges in order to keep all alleles less than 350 bp through careful examination of known allele ranges. Haplotype comparisons between the 20plex and a commercially available kit found excellent agreement across the 76 samples in the Y chromosome consortium panel.     

A collaborative study of the EDNAP group regarding Y-chromosome binary polymorphism analysis

A collaborative study was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the performance of Y-chromosome binary polymorphism analysis in different European laboratories. Four blood samples were sent to the laboratories, to be analysed for 11 Y-chromosome single nucleotide polymorphisms (SNPs): SRY-1532, M40, M35, M213, M9, 92R7, M17, P25, M18, M153 and M167. All the labs were also asked to submit a population study including these markers. All participating laboratories reported the same results, indicating the reproducibility and robustness of Y-chromosome SNP typing. A total of 535 samples from six different European populations were also analysed. In Galicia (NW Spain) and Belgium, the most frequent haplogroup was R1b*(xR1b1,R1b3df). Haplogroup F*(xK) is one of the most frequent in Austria and Denmark, while the lowest frequency appear in Belgium. Haplogroup frequencies found in this collaborative study were compared with previously published European Y-chromosome haplogroup data.     

Y chromosome J2 subtyping in an Italian sample: Population and forensic implications

In the recent years the Y chromosome genealogy has been refined by a number of newly discovered SNPs. The non-random distribution of the Y chromosome lineages worldwide makes fundamental the dissection and characterisation of haplogroups associated with specific geographic areas. In Southern Europe the haplogroup J2, as defined by the M172 marker, can reach frequencies up to 35%, making the dissection of such lineage critical for population studies. Here we present a study on J2 chromosomes from the Italian peninsula. Populations and forensic implications are discussed. A total of 900 individuals were previously genotyped for a number of SNPs, including M172. More than 200 of these have been now genotyped for 7 SNPs within the J2 lineage using a multiplex SNaPshot approach. The different distribution of the various lineages in different geographic areas probably reflects different historical demographic events and points to differential Y chromosome haplotype distribution, with implication for forensic application of this genetic marker.     

Robustness of the Y STRs DYS19, DYS389 I and II, DYS390 and DYS393: optimization of a PCR pentaplex

Various technical methods were investigated with the aim of developing a multiplex system to amplify five Y-chromosome STR loci in the same PCR reaction: DYS393, DYS19, DYS390, DYS389 I and DYS389 II. A sequenced allelic ladder was constructed with previously sequenced alleles including the most common ones. A number of reamplification conditions of the allelic ladders were tested. The pentaplex was evaluated for typing using two different platforms (ABI and ALF) with promising results. However, in degraded samples non-specific artifacts were observed in the DYS393 system in the same range of sizes as the real alleles. This system can also be typed in females under relatively low stringency conditions in the PCR amplification, making this system prone to errors in critical samples. This lack of specificity can be reduced by increasing the stringency of the PCR conditions. The DYS19 ladder cannot be reamplified as stutters appear after a few reamplifications. These stutters are probably due to a 2 bp slippage induced by the presence of a TA repeat stretch in the PCR amplified fragments. Non-specific products were also noted in the DYS389 I and DYS389 II amplification, although out of the range of other alleles in this pentaplex. This newly constructed pentaplex has proved to be very useful in population genetic studies because all five Y STR markers can be loaded in the same lane of a gel with other Y STR singleplex or multiplexes. The usefulness of Y-chromosome STRs in criminal casework is especially evident in analyzing azoospermic individuals.     

Microgeographic genetic variation of Y chromosome in a population sample of Ravenna's area in the Emilia-Romagna region (North of Italy)

The analysis of the genetic structure of regions with a complex demographic history shed light on the various topographic, linguistic and historical influences which form the present genetic landscape of Europe. In the Emilia-Romagna region (North of Italy) Ravenna is a geographical area with a historical complex background: it was an important seaport on the Mediterranean sea, the capital of the Ostrogothic kingdom of Italy and the seat of the Byzantine governor of Italy. The purpose of this study was to investigate the microgeographic variation of Y chromosome haplotypes of the area of Ravenna by analyzing 17 Y-chromosome short tandem repeats (Y-STRs) in 122 unrelated males. 100% of all haplotypes were different. A comparison with neighbouring Italian as well as with European and Levante root populations was done by AMOVA and visualized by a phylogenetic tree. The two main haplogroups found in this area were R1b and E3b1. The results of the present study add to the data for the forensic databases and can be useful also for anthropological studies.     

Multiplex PCR and minisequencing of SNPs--a model with 35 Y chromosome SNPs

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.     

Developmental validation of 15 X chromosomal short tandem repeat markers

The use of X chromosomal short tandem repeat (STR) markers has been greatly increasing in the forensic setting. Using guidelines set forth previously for the validation of autosomal and Y STRs, aspects of the feasibility of routine X chromosomal STR use were evaluated. Two mini-X chromosomal STR multiplexes capable of amplifying 15 total markers were developed and utilized to determine allele nomenclature, allele/genotype frequencies, mutation rates, and linkage between markers. Additionally, a concordance study between these multiplexes and a commercially available kit was performed. Here, the authors present an overview of this extensive developmental validation study.     

Validation and casework testing of the BioPlex-11 for STR typing of telogen hair roots

A new STR typing strategy has been developed allowing the simultaneous amplification and subsequent analysis of 11 polymorphic systems with amplicon sizes smaller than 270bp. The multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THO1, FGA and VWA) as well as four additional STR systems selected for their robustness (D2S1338, D12S391, TPOX and D5S818) together with the sex-specific locus amelogenin. After PCR amplification, the multiplex reaction is splitted into two sets of STR multiplexes by using biotin labelled primers only for one set. Using streptavidin-coated Sepharose beads five STR systems are separated from the other six systems prior to being analysed in two different runs on a capillary gel electrophoresis instrument. The multiplex system was developed and tested especially for the use in forensic casework if only limited amounts or highly degraded DNA is available, for instance, when isolated from telogen hair roots.     

The use of the LightCycler for the detection of Y chromosome SNPs

A novel methodology based on PCR monitoring on-line with fluorescent formats using the LightCycler for Y chromosome SNP typing is proposed. The main advantages of the system are the time necessary for the analysis (which is around 20 min), the robustness and the accuracy of the method and especially its sensitivity, which permits the detection of the male component in male-female mixtures up to 1:300 for some of the SNPs.Singleplexes of four different SNPs (M9, sY81, SRY-1532 and SRY-2627) as well as two duplexes (M9 and sY81 on the one hand and SRY-1532 and SRY-2627 on the other) were efficiently implemented. A simultaneous amplification and analysis of the four SNPs is also possible. It seems difficult with the current methodology to implement more than a quadruplex.     

二代测序技术在Y染色体遗传标记分型中的法医学应用

法医遗传学领域常利用Y染色体的父系遗传特点,对非重组区遗传标记进行检测并用于亲缘关系鉴定、混合斑检测、家系排查以及种族推断等研究。目前毛细管电泳仍是应用最为广泛的检测技术,基于该技术的商业化检测试剂盒及数据分析处理系统十分成熟。随着生物信息量的增长,传统检测技术通量低的弊端逐渐显现,推动了法医DNA分型技术的革新。近年来,二代测序(next generation sequencing,NGS)技术发展迅速,其应用已被推广到包括法医遗传学在内的各领域,为Y染色体遗传标记的检测提供了新的技术手段。本文就NGS技术应用于法医学Y染色体遗传标记检测的研究现况和应用前景进行阐述,以期为后续司法实务提供新思路。