福建畲族人群15个短串联重复序列的遗传多态性及法医学应用

目的对福建畲族群体15个短串联重复序列等位基因遗传多态性进行分析,探讨其种族特异性及其在法医学个体识别和亲权鉴定的中的应用。方法荧光标记15个短串联重复序列(STR)位点和一个性别位点并进行PCR复合扩增,用ABI遗传分析仪以毛细管电泳技术对100例福建畲族无血缘关系健康个体PCR产物进行电泳分离基因,并以分析软件进行基因分型。结果共检出126个等位基因,基因频率范围在0.0050～0.5550。15个STR基因座的基因型分布均符合Hardy-Weinberg平衡。15个STR位点的HET在0.6300～0.8900之间,DP在0.7680～0.9590之间,EPP在0.3280～0.7750之间,PIC在0.5200～0.8600之间。累积DP和EPP分别达到0.99999999和0.99999288。结论15个STR位点均有较高的遗传多态性,并有丰富的信息含量,适合作为福建畲族地区的遗传标记,可用于个体识别和亲权鉴定。     

Assessing non-LUS stutter in DNA sequence data

Forensic DNA analysis is among the most well-recognized and well-developed forensic disciplines. The field’s use of DNA markers known as short tandem repeats (STRs) offer a robust means of discriminating individuals while also introducing challenges to the analysis. One of these challenges, stutter, is the result of a non-biological artifact introduced during PCR. The formation and amplification of these stutter products can occur at rates as high as 15–20% of the parent allele. The challenge inherent in this process is differentiating stutter artifacts from true alleles, particularly in the presence of a minor contributor. Traditionally, DNA profiles are obtained using capillary electrophoresis (CE), where amplified DNA fragments are separated by size, not sequence, and the identification of stutter is performed on a locus-specific level. The use of CE-based fragment data rather than sequence-based data, has limited the community’s understanding of the precise behavior of stutter. Massively parallel sequencing (MPS) data provides an opportunity to better characterize stutter, permitting a more accurate means of detecting both size- or longest uninterrupted stretch (LUS)-based stutter but also allele and motif-specific stutter characteristics. This study sheds light on the value of characterizing motif- and allele-specific stutter, including non-LUS stutter, when using MPS methods. Analysis and characterization of stutter sequences was performed using data generated from 539 samples amplified with the ForenSeq and PowerSeq 46GY library preparation kit and sequenced on the Illumina MiSeq FGx. Assessment of non-LUS stutter begins with calculating stutter rates for all potential stutter products at a given locus (and allele), additionally, the occurrence of these discrete stutter products were quantified. Results show that although the LUS sequence stutters at a higher rate than non-LUS motifs, the non-LUS stutter products do occur at detectable levels and potentially influence sequence-based mixture analysis. The data indicate that the stutter from one motif or allele can be distinguished from another motif or allele based on their unique stutter rates; however, the number of stutter products from each motif or allele may similarly make up the overall pool of stutter products. Motif- and allele-specific stutter models provide the most comprehensive analysis of sequence stutter rates and provide the ability to differentiate stutter sequences more accurately from true allele stutter. This information provides a foundation for including the characterization of non-LUS stutter products when analyzing DNA profiles, specifically mixtures with potential low-level contributors.     

A population study of the STR loci HUMLPL, HUMF13B, and HUMF13A01 in Hungary

Allele frequencies of the three STR systems HUMLPL, HUMF13B, and HUMF13A01 were determined in a Hungarian population sample of 223 unrelated Caucasian individuals. All loci met Hardy-Weinberg expectations and there was no evidence for association of alleles among the three STR loci. In addition, little evidence was found for departures from expectations of independence between any of the three STR and other previously investigated microsatellite polymorphisms. Received: 19 October 1996 / Received in revised form: 6 January 1997     

重庆土家族群体15个STR基因座的遗传多态性

目的:研究重庆土家族人群无关个体的15个STR基因座(CSF1PO、D13S317、D16S539、D18S51、D19S433、D21S11、D2S1338、D3S1358、D5S818、D7S820、D8S1179、FGA、TH01、TPOX、vWA)的遗传多态性.方法:应用AmpFlSTR Identifiler荧光标记复合扩增系统对115名重庆土家族无关个体血样DNA进行15个STR基因座的复合扩增,用ABI310遗传分析仪对扩增产物进行检测,用GeneScan、GenoTyper软件进行基因分型,统计计算15个STR基因座的群体遗传学参数.结果:15个STR基因座均符合Hardy-Weinberg平衡,杂合度在0.634 8～0.887 0之间,累积匹配概率和累积非父排除率分别为3.12×10-17和0.999 997 688 3.结论:15个STR基因座的累积匹配概率和累积非父排除率较高,适用于法医学亲子鉴定和个人识别.     

High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6–200 ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62 pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250 pg of input DNA, and 62 pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.     

An optimized procedure for obtaining DNA from fired and unfired ammunition

Gun crimes are a significant problem facing law enforcement agencies. Traditional forensic examination of firearms involves comparisons of markings imparted to bullets and cartridge casings during the firing process. DNA testing of casings and cartridges may not be routinely done in crime laboratories due a variety of factors including the typically low amounts of DNA recovered. The San Diego Police Department (SDPD) Crime Laboratory conducted a study to optimize the collection and profiling of DNA from fired and unfired ammunition. The method was optimized to where interpretable DNA results were obtained for 26.1% of the total number of forensic casework evidence samples, and provided some insights into the level of secondary transfer that might be expected from this type of evidence. Briefly detailed are the results from the experimental study and the forensic casework analysis using the optimized process. Mixtures (samples having more DNA types than the loader’s known genotype detected or visible at any marker) were obtained in 39.8% of research samples and the likely source of DNA mixtures is discussed.     

Characterization of 114 insertion/deletion (INDEL) polymorphisms,and selection for a global INDEL panel for human identification

Bi-Allelic Insertions and Deletions (INDELs) are a powerful set of genetic markers for Human Identification (HID). They have certain desirable features, such as low mutation rates, no stutter, and potentially small amplicon sizes that could prove effective in some circumstances. In this study, we analyzed the distribution of 114 INDELs in four North American populations (Caucasian, African American, Southwest Hispanic, and Asian) to estimate their distribution in major global populations. Of the 114 INDELs a primary panel of 38 candidate markers was selected that met the criteria of (1) a minimum allele frequency of greater than 0.20 across the populations studied; (2) general concordance with Hardy–Weinberg equilibrium (HWE) expectations; (3) relatively low F_ST based on the major populations; (4) physical distance between markers greater than 40 Mbp; and (5) a lack of linkage disequilibria between syntenic markers. Additionally, another 11 supplemental markers were selected for an expanded panel of 49 markers which met the above criteria, with the exception that they are separated at least by 20 Mbp. The resulting panels had Random Match Probabilities that were at least 10⁻¹⁶ and 10⁻¹⁹, respectively, and combined F_ST values of approximately 0.02. Given these findings, these INDELs should be useful for HID.     

短串联重复序列基因座在石蜡包埋组织中的应用

目的探讨3种STR基因座分型系统在石蜡包埋组织基因座分型的差异。方法采用Qiagen法对保存1个月的石蜡包埋组织进行DNA提取,用Identifiler系统、PowerPlex 21系统及Investigator HDplex系统对STR基因座进行聚合酶链反应复合扩增、毛细管电泳、荧光检测及片段分析,比较3种系统的STR基因座检出率,并且采集组织同一个体的血液、毛发、口腔拭子等样本进行STR基因座分型,比较同一个体不同器官组织STR基因座分型的差异。结果经紫外分光光度计测定石蜡包埋组织的DNA浓度为6~85ng/μL,纯度1.7~2.2,Identifiler系统能够在DNA浓度大于15ng/μL时得到完整的基因座分型,PowerPlex 21系统在DNA浓度为85ng/μL时得到完整的基因座分型,而HDplex系统在石蜡组织检测中均未获得完整的基因座分型。石蜡包埋组织与其他器官组织分型结果存在差异,其中D6S474、D4S2366和D21S2055基因座存在等位基因不平衡或基因座丢失现象。结论石蜡包埋组织中DNA的浓度和纯度是STR基因座分型的重要影响因素,遗传信息丢失现象与检验对象的性状有关,也与所采用的检测系统有关,Identifiler系统对石蜡包埋组织的STR基因座分型具有较高的实用价值。     

优化甲醛固定石蜡包埋组织DNA提取预处理对STR分型有效性的研究

目的通过对甲醛固定石蜡包埋组织（FFPET）DNA提取进行优化处理,确定满足FFPET样本进行短片段重复序列（STR）完整分型的条件。方法改变石蜡切片厚度、离心速率和时间、组织用量和消化液体积等参数提取FFPETDNA,并对提取的DNA样本进行常规STR扩增检验。结果优化方法处理后可以提取到较高质量DNA（1000bp）,经扩增均可以检出性别基因座（amelogenin）和15个以上STR基因座。结论 FFPET优化方法能够提高DNA提取的质量,适合STR分型研究。