American Registry of Pathology, Camden, DE, United States; Armed Forces DNA Identification Laboratory, Armed Forces Medical Examiner System, Dover AFB, DE, United States.
American Registry of Pathology, Camden, DE, United States; Armed Forces DNA Identification Laboratory, Armed Forces Medical Examiner System, Dover AFB, DE, United States.
Forensic Sci Int Genet. 2014 Sep;12:107-19. doi: 10.1016/j.fsigen.2014.04.010. Epub 2014 May 4.
To investigate the feasibility of next generation sequencing technology (NGS) for the multiplex detection and sequence production of short tandem repeats (STRs) from degraded and low DNA quantity samples, standard polymerase chain reaction amplification methods were used to enrich for commonly employed STR markers. Samples were amplified with two multiplexing strategies: a multiplex containing thirteen miniSTR markers and a series of multiplexes containing four miniSTR markers each. Each sample multiplex was barcoded with a sample-specific multiplex identifier for subsequent parallel tagged sequencing on the GS Junior System (454 Life Sciences, a Roche company, Branford, CT). Sequencing results from over fifty DNA extracts representing both pristine samples and low-quality evidentiary specimens reflected known genotypes and were consistent across multiple extracts and/or amplifications of the same sample. Furthermore, the NGS data revealed sequence information not available with standard capillary electrophoresis-based detection alone. For the population samples tested, a total of 152 single nucleotide polymorphisms or insertions/deletions were identified in over 935 recovered alleles, averaging one polymorphism for every six recovered alleles. For three of the loci, the sequence information doubled the number of alleles detected via traditional STR typing by fragment analysis. In addition, twenty-eight of these variants were only seen once within our dataset, highlighting the potential for discrimination among individuals. These additional data are likely to be particularly valuable in missing persons and disaster victim identification cases for which only partial profiles may be recovered and/or only distant kin are available as references. And, considering the opportunity to target only small amplicons with NGS, this type of STR typing will allow for greater information recovery from challenging casework samples. While our results highlight the potential of new technologies for recovering discriminatory genetic information from evidentiary specimens, our data also reveal the complexities of NGS-based STR typing, both in terms of the laboratory assays themselves as well as the downstream data processing and analysis.
为了研究下一代测序技术（NGS）在从降解和低 DNA 量样品中多重检测和序列产生短串联重复序列（STRs）的可行性，使用标准聚合酶链反应扩增方法来富集常用的 STR 标记物。使用两种多重化策略对样品进行扩增：包含 13 个微 STR 标记物的多重化和包含每个 4 个微 STR 标记物的一系列多重化。每个样品多重化都用样品特异性多重化标识符进行标记，以便随后在 GS Junior 系统（454 Life Sciences，Roche 公司，Branford，CT）上进行并行标记测序。代表原始样品和低质量证据样本的五十多个 DNA 提取物的测序结果反映了已知的基因型，并且在多个提取物和/或相同样品的多次扩增中是一致的。此外，NGS 数据揭示了仅用标准毛细管电泳检测无法获得的序列信息。对于测试的人群样本，在超过 935 个回收的等位基因中总共鉴定出 152 个单核苷酸多态性或插入/缺失，平均每个回收等位基因有一个多态性。对于三个基因座，序列信息使通过传统 STR 分型的片段分析检测到的等位基因数量增加了两倍。此外，这些变体中的 28 个仅在我们的数据集中出现过一次，突出了个体之间区分的潜力。这些额外的数据可能在失踪人员和灾难受害者识别案例中特别有价值，因为可能只回收了部分图谱，或者只有远亲可以作为参考。而且，考虑到有机会仅用 NGS 靶向小扩增子，这种类型的 STR 分型将允许从具有挑战性的案例工作样本中获得更多信息。虽然我们的结果强调了新技术从证据样本中恢复有区别的遗传信息的潜力，但我们的数据也揭示了基于 NGS 的 STR 分型的复杂性，包括实验室检测本身以及下游数据处理和分析。
