AB1 chromatograms used to validate the ConTAMPR method for detecting DNA contamination

Contingency Table Analysis of Minority Peak Ranks (ConTAMPR) is a method to interrogate Sanger sequencing chromatograms for evidence of multiple substantially divergent DNA sequences, and to identify a contaminant statistically from a panel of candidates. It was originally developed and used to test for cross-contamination in PCR amplicons generated from samples of DNA extracted from bdelloid rotifers in the genus Adineta. The 38 chromatograms in this dataset were generated during an experiment in which two rotifers of different species were deliberately placed in the same tube and had their DNA extracted together. A fragment of the mitochondrially encoded cytochrome c oxidase subunit I was amplified by PCR using the primers HCO1 and LCO1 and the amplicons were sequenced in both directions by Macrogen Europe on an ABI 3730xl DNA Analyzer (Applied Biosystems). Samples in the "1X" groups had DNA from a single rotifer (either Adineta vaga AD008 or A. sp. AD006). Samples in the "2X" group had DNA from two rotifers, one of each species. Multiple biological replicates are indicated with numbers (01-06), and technical replicates with letters (a-c). The chromatograms for "1X" samples have some noise (small minority peaks, base call errors, etc.), but this noise is not systematic. Each of the two species has a characteristic and consistent mitochondrial haplotype. The chromatograms for "2X" samples also each show a majority haplotype corresponding to one of the two species, but the identity of this majority haplotype differs from sample to sample, and the second species is revealed via a systematic pattern of minority fluorescence peaks at sites where the two species differ. This pattern can be analysed by focusing on sites where the two species differ, and looking for the minority fluorescence peaks corresponding to the predicted contaminant haplotype. Each peak is assigned a height rank (2, 3 or 4). The contaminant can be identified statistically by tabulating these peak ranks and using a Chi-square contingency test. The contaminant haplotype will show a statistically significant excess of second-ranked peaks relative to a null (equal) expectation and also relative to other candidate contaminants. This method was validated using eight of the files in this dataset: LCO1_2X_6_8_05a; HCO1_2X_6_8_05a; LCO1_2X_6_8_03a; HCO1_2X_6_8_03a; LCO1_1X_6_01a; HCO1_1X_6_01a; LCO1_1X_8_02c; HCO1_1X_8_02c . Biological and technical replicates showed comparable patterns. For further details please consult the associated publication.少数峰秩列联表分析法（ConTAMPR）是一种用于分析桑格测序（Sanger sequencing）色谱图，以检测存在多种显著差异DNA序列的证据，并从候选序列组中统计学鉴定污染物的方法。该方法最初开发并用于检测从Adineta属蛭形轮虫样本中提取DNA后生成的PCR扩增产物（PCR amplicons）的交叉污染情况。本数据集包含38份色谱图，源自一项实验：将两种不同物种的轮虫置于同一试管中共同提取DNA。通过引物HCO1和LCO1进行PCR扩增线粒体编码的细胞色素c氧化酶亚基I（mitochondrially encoded cytochrome c oxidase subunit I）片段，扩增产物由Macrogen Europe公司在ABI 3730xl DNA分析仪（Applied Biosystems）上进行双向测序。 “1X”组样本的DNA来自单只轮虫（分别为Adineta vaga AD008或A. sp. AD006）；“2X”组样本的DNA来自两种各一只的轮虫。生物学重复（biological replicates）以数字（01-06）标注，技术重复（technical replicates）以字母（a-c）标注。“1X”样本的色谱图存在一定噪声（少量少数峰、碱基识别错误（base call errors）等），但该噪声不具有系统性。两个物种各自拥有特征性且一致的线粒体单倍型（haplotype）。“2X”样本的色谱图均显示出对应两种物种之一的主要单倍型，但不同样本的主要单倍型种类存在差异，且在两个物种存在序列差异的位点处，可通过系统性的少数荧光峰模式揭示第二个物种的存在。 该分析模式可通过聚焦于两物种存在序列差异的位点，并寻找与预测污染物单倍型对应的少数荧光峰来开展。每个峰会被赋予高度秩次（2、3或4）。通过对这些峰秩进行列联表制表，并采用卡方列联检验（Chi-square contingency test），可统计学鉴定污染物。与零假设（等概率期望）及其他候选污染物相比，污染物单倍型会在第二秩峰的数量上表现出统计学显著的过量。 本研究使用数据集中的8份文件对该方法进行了验证：LCO1_2X_6_8_05a、HCO1_2X_6_8_05a、LCO1_2X_6_8_03a、HCO1_2X_6_8_03a、LCO1_1X_6_01a、HCO1_1X_6_01a、LCO1_1X_8_02c、HCO1_1X_8_02c。生物学重复与技术重复展现出相似的分析模式。更多细节可查阅相关研究论文。