Interlaboratory study to validate a STR profiling method for intraspecies identification of mouse cell lines

The Consortium for Mouse Cell Line Authentication was formed to validate Short Tandem Repeat (STR) markers for intraspecies identification of mouse cell lines. The STR profiling method is a multiplex polymerase chain reaction (PCR) assay comprised of primers targeting 19 mouse STR markers and two human STR markers (for interspecies contamination screening). The goals of the Consortium were to perform an interlaboratory study to–(1) validate the mouse STR markers to uniquely identify mouse cell lines (intraspecies identification), (2) to provide a public database of mouse cell lines with the National Institute of Standards and Technology (NIST)-validated mouse STR profiles, and (3) to publish the results of the interlaboratory study. The interlaboratory study was an international effort that consisted of 12 participating laboratories representing institutions from academia, industry, biological resource centers, and government. The study was based on 50 of the most commonly used mouse cell lines obtained from the American Type Culture Collection (ATCC). Of the 50 mouse cell lines, 18 had unique STR profiles that were 100% concordant (match) among all Consortium laboratory members, and the remaining 32 cell lines had discordance that was resolved readily and led to improvement of the assay. The discordance was due to low signal and interpretation issues involving artifacts and genotyping errors. Although the total number of discordant STR profiles was relatively high in this study, the percent of labs agreeing on allele calls among the discordant samples was above 92%. The STR profiles, including electropherogram images, for NIST-validated mouse cell lines will be published on the NCBI BioSample Database (https://www.ncbi.nlm.nih.gov/biosample/). Overall, the interlaboratory study showed that the multiplex PCR method using 18 of the 19 mouse STR markers is capable of discriminating at the intraspecies level between mouse cell lines. Further studies are ongoing to refine the assay including (1) development of an allelic ladder for improving the accuracy of allele calling and (2) integration of stutter filters to identify true stutter.小鼠细胞系鉴定联盟（Consortium for Mouse Cell Line Authentication）成立的宗旨，是验证用于小鼠细胞系种内鉴定的短串联重复序列（Short Tandem Repeat, STR）标记。该STR分型技术为多重聚合酶链式反应（polymerase chain reaction, PCR）检测体系，其引物靶向19个小鼠STR标记与2个人类STR标记，用于种间污染筛查。该联盟的目标包括开展实验室间研究以达成三项任务：(1) 验证小鼠STR标记，以实现小鼠细胞系的唯一标识（种内鉴定）；(2) 搭建公开的小鼠细胞系数据库，收录经美国国家标准与技术研究院（National Institute of Standards and Technology, NIST）验证的小鼠STR分型数据；(3) 发表该实验室间研究的成果。本次实验室间研究为国际性合作项目，共有12家来自学术界、工业界、生物资源保藏机构及政府部门的实验室参与。研究依托从美国典型培养物保藏中心（American Type Culture Collection, ATCC）获取的50株最常用小鼠细胞系展开。在这50株小鼠细胞系中，18株的STR分型结果在联盟所有实验室间完全一致（匹配度100%）；剩余32株的分型结果存在不一致，但该问题可被顺利解决，并推动了检测体系的优化。此类不一致源于信号强度不足以及涉及伪峰与基因分型错误的判读问题。尽管本研究中STR分型结果不一致的总占比相对较高，但在存在不一致的样本中，各实验室对等位基因判读的一致率仍超过92%。经NIST验证的小鼠细胞系的STR分型数据（包括电泳峰图图像）将发表于NCBI生物样本数据库（https://www.ncbi.nlm.nih.gov/biosample/）。总体而言，本次实验室间研究证实，使用19个小鼠STR标记中的18个所构建的多重PCR体系，可实现小鼠细胞系的种内区分。目前相关优化研究正在进行中，包括(1) 开发等位基因阶梯以提升等位基因判读的准确性；(2) 集成伪峰过滤模块以识别真实的扩增伪峰。