滇西亚种树鼩微卫星分子标记的筛选

目的 筛选出适用于滇西亚种树鼩种群遗传质量控制的特异性微卫星分子标记。方法 首先从树鼩全基因组序列中筛选出约700个微卫星位点, 择优选出约100个位点设计引物, 去除有不良因素的, 最后保留33对和文献报道的13对引物对滇西亚种树鼩DNA进行 PCR扩增, 根据琼脂糖电泳和聚丙烯酰胺电泳结果筛选保留, 进行STR扫描再次筛选适于树鼩遗传检测的微卫星位点组合。结果 筛选出树鼩微卫星位点22个, STR基因扫描有明显Stutter峰。比较备选位点和最终筛选出的位点, 普通树鼩位点中选取5个, 2个可用, 与滇西亚种的重合率是40%;印度小树鼩位点中选取5个, 2个可用, 重合率是40%;中缅树鼩的位点中选取3个, 2个可用, 重合率约70%。结论 筛选出的22个微卫星位点适用于滇西亚种树鼩的遗传检测, 为树鼩种群的遗传质量监测提供科学依据。     

What’s on the bag? The DNA composition of evidence bags pre- and post-exhibit examination

Current forensic DNA profiling kits and techniques enable the detection of trace amounts of DNA. With advancements in kit sensitivity, there is an increased probability of detecting DNA from contamination. Research into DNA transfer within operational forensic laboratories provides insight into the possible mechanisms that may lead to exhibit contamination. To gain a greater understanding of the potential for evidence bags to act as DNA transfer vectors, the level of DNA accumulating on the exterior of evidence bags during the exhibit examination process was investigated. The exterior of 60 evidence bags were tapelifted before and after the examination of the exhibit inside of the bag resulting in 120 DNA profiles. These DNA profiles were compared to DNA profiles of staff working within the building and samples taken from the exhibit inside the bag. Common DNA profile contributors from each sample were also identified through STRmix™ mixture to mixture analysis. The average DNA quantity and number of profile contributors was higher in samples taken from the bag before exhibit examination than after examination. Fifty six percent of all samples taken identified a match between DNA recovered from the evidence bag and at least one staff member. On 11 bags, a common contributor was identified between the exhibit in the bag and the exhibit package post-examination. In one instance a DNA profile, matching that of a donor, on the exhibit bag before examination was also detected on a sample taken from the exhibit, raising the possibility of outer bag-to-exhibit DNA contamination. This study demonstrates that operational forensic laboratories must consider exhibit packages as a potential source of DNA contamination and evaluate their exhibit handling and storage procedures accordingly.     

Exploring tapelifts as a method for dual workflow STR amplification

Although a version of direct PCR is implemented in forensic laboratories for reference material, its incorporation into workflow for the analysis of touch DNA, as a form of latent DNA, from casework exhibits is not. In addition to concerns about increased sensitivity causing more complex mixtures or the generation of more genetic data implicating an individual superfluous to the context of the alleged event, the complete use of the collected sample in the PCR as template has meant that there is no possibility for data reproducibility when needed. Here it is proposed that the use of tapelifts in touch DNA collection can facilitate replicate direct PCR analysis from a single sample allowing the sample to be re-tested. If all portions of the tapelift result in profiles with allelic and likelihood ratio concordance, these sub-samples may be accepted as technical replicates, thus meeting any accreditation guideline requirements. Furthermore, we assess the use of a single tapelift for both direct PCR and extraction-based PCR workflows to illustrate the potential for benefits of both systems to be facilitated. DNA was deposited by three donors onto six substrates with five sample replicates of each condition. Separation of each tapelift into three portions for three direct PCRs ensued using VeriFiler™ Plus. Separation of single tapelifts into three direct PCRs showed no statistical difference in donor allele calls or RFU, or subsequent LRs associated with their profiles. Comparison of profiles within the single tapelift showed more similarity, with high mixture-to-mixture match likelihoods, than when these sub-samples were compared with profiles generated from other samples. This allows each sub-sample taken from the tapelift to be considered as technical replicates. For dual workflow facilitation assessment, one donor deposited DNA through touch onto six substrates with five research replicates of each. Separation of single tapelifts into two portions, one for direct PCR and the retention and use of the remaining portion for extraction and subsequent PCR, showed no significant difference in allelic yield and subsequent donor comparison LRs. Comparison of deconvoluted profiles produced from a single tapelift showed high mixture-to-mixture match likelihoods, supporting DNA donor concordance. This indicates that removing a portion of a tapelift for direct PCR amplification, while processing the remainder through standard processes, allows increased sensitivity through direct PCR while offering the preparation of an eluate suitable for repeated analyses.     

Ion Torrent ™ Genexus ™ Integrated Sequencer and ForeNGS Analysis Software—An automatic NGS-STR workflow from DNA to profile for forensic science

The Ion Torrent ™ Genexus ™ Sequencer (Genexus) is a highly integrated instrument that can automate library construction, templating, and sequencing in a single-instrument run. By programing the ForeNGS Analysis Software (FNAS), we bridged the gap between sequencing and genotyping without manual intervention. FNAS can automatically transfer sequencing output files from Genexus, analyze the repeat and flanking regions aligned to the GRCh38 assembly, name the alleles according to the ISFG guidelines, and generate user-friendly interactive profiles. Genexus and FNAS can accomplish the fully automatic DNA-to-Profile workflow in forensics. Based on our experiences, the optimal assay parameters on Genexus were validated as follows: 24 cycles of target amplification for library construction; 40 μL of library and 400 bp of template size for templating; 852 flows of dNTPs by order of Ion samba HID2 for sequencing; and 750,000 reads per sample at minimum for 16 samples multiplexed on a lane. By developmental validations of the Precision ID Globalfiler ™ NGS STR Panel v2, Genexus presented competitive performance at the optimal assay parameters qualified to detect commonly used forensic STR markers. It could produce repeatable and reproducible results, and human profiles could be easily separated from nonhuman profiles. Additionally, Genexus was sensitive enough to detect samples with 100 pg of input DNA, and it was suitable for various types of case samples, especially for low copy number samples and degraded samples. Moreover, minor contributors could be detected between the 4:1 and 1:4 mixtures with an analysis threshold of 50 × . The Genexus workflow is a robust and labor-effective solution enabling forensic scientists to obtain NGS-STR profiles within a single day and with only the need to prepare DNA extracts, then set up Genexus, and finally interpret profiles on FNAS.     

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.     

Genetic polymorphisms of 15 STR loci in the population of the island of Cres (Croatia)

Background: The population of the island of Cres presents one of the few persisting Eastern Adriatic isolates and is thereby suitable for human population differentiation analyses.

Aim: The aim of this study was to analyse the genetic structure of the island of Cres with respect to its eight sub-populations and to compare the genetic variation of the island of Cres with other Eastern Adriatic islands and the Croatian mainland.

Subjects and methods: Fifteen AmpFlSTR identifiler loci were analysed in a sample group of 122 unrelated autochthonous individuals from the island of Cres, Croatia.

Results: Analysis of STR polymorphisms revealed genetic homogeneity among sub-populations of the island of Cres and small but significant levels of genetic heterogeneity among geographically distant Eastern Adriatic islands.

Conclusion: Despite a considerable degree of genetic homogeneity among the studied Eastern Adriatic islands, small but significant differentiation between distant islands indicates geographic sub-structuring which follows the isolation by distance model. This study is supportive of the notion that STR markers are useful for genetic differentiation between larger and geographically more distant regions.     

Amelogenin test abnormalities revealed in Belarusian population during forensic DNA analysis

Study of gender markers is a part of routine forensic genetic examination of crime scene and reference samples, paternity testing and personal identification. Amelogenin locus as a gender marker is included in majority of forensic STR kits of different manufacturers. In current study we report 11 cases of amelogenin abnormalities identified in males of Belarusian origin: 9 cases of AMELY dropout and 2 cases of AMELX dropout. Cases were obtained from forensic casework (n = 9) and paternity testing (n = 2) groups. In 4 out of 9 AMELY-negative cases deletion of AMELY was associated with the loss of DYS458 marker. In addition, we identified 3 males with SRY-positive XX male syndrome. Deletion of the long arm of the Y-chromosome was detected in two XX males. Loss of the major part of the Y-chromosome was identified in the third XX male. The presence of two X-chromosomes in XX males was confirmed with the use of Mentype^® Argus X-8 PCR Amplification Kit. AMELY null allele observed in 2 out of 9 cases with AMELY dropout can be caused by mutation in the primer-binding site of AMELY allele. Primer-binding site mutations of AMELX can result in AMELX dropout identified in 2 cases with amplification failure of AMELX. Our study represents the first report and molecular genetic investigation of amelogenin abnormalities in the Belarusian population.     

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。