靶向 RNAseq 分析结合独特分子标识符，可提高固定肿瘤样本中基因表达特征和转录突变分数的定量分析。
Targeted RNAseq assay incorporating unique molecular identifiers for improved quantification of gene expression signatures and transcribed mutation fraction in fixed tumor samples.
机构信息
Departments of Pathology and Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.
出版信息
BMC Cancer. 2021 Feb 4;21(1):114. doi: 10.1186/s12885-021-07814-8.
BACKGROUND
Our objective was to assess whether modifications to a customized targeted RNA sequencing (RNAseq) assay to include unique molecular identifiers (UMIs) that collapse read counts to their source mRNA counts would improve quantification of transcripts from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. The assay (SET4) includes signatures that measure hormone receptor and PI3-kinase related transcriptional activity (SET and PI3Kges), and measures expression of selected activating point mutations and key breast cancer genes.
METHODS
Modifications included steps to introduce eight nucleotides-long UMIs during reverse transcription (RT) in bulk solution, followed by polymerase chain reaction (PCR) of labeled cDNA in droplets, with optimization of the polymerase enzyme and reaction conditions. We used Lin's concordance correlation coefficient (CCC) to measure concordance, including precision (Rho) and accuracy (Bias), and nonparametric tests (Wilcoxon, Levene's) to compare the modified (NEW) SET4 assay to the original (OLD) SET4 assay and to whole transcriptome RNAseq using RNA from matched fresh frozen (FF) and FFPE samples from 12 primary breast cancers.
RESULTS
The modified (NEW) SET4 assay measured single transcripts (p< 0.001) and SET (p=0.002) more reproducibly in technical replicates from FFPE samples. The modified SET4 assay was more precise for measuring single transcripts (Rho 0.966 vs 0.888, p< 0.01) but not multigene expression signatures SET (Rho 0.985 vs 0.968) or PI3Kges (Rho 0.985 vs 0.946) in FFPE, compared to FF samples. It was also more precise than wtRNAseq of FFPE for measuring transcripts (Rho 0.986 vs 0.934, p< 0.001) and SET (Rho 0.993 vs 0.915, p=0.004), but not PI3Kges (Rho 0.988 vs 0.945, p=0.051). Accuracy (Bias) was comparable between protocols. Two samples carried a PIK3CA mutation, and measurements of transcribed mutant allele fraction was similar in FF and FFPE samples and appeared more precise with the modified SET4 assay. Amplification efficiency (reads per UMI) was consistent in FF and FFPE samples, and close to the theoretically expected value, when the library size exceeded 400,000 aligned reads.
CONCLUSIONS
Modifications to the targeted RNAseq protocol for SET4 assay significantly increased the precision of UMI-based and reads-based measurements of individual transcripts, multi-gene signatures, and mutant transcript fraction, particularly with FFPE samples.
背景
我们的目标是评估对定制的靶向 RNA 测序（RNAseq）检测进行修改，包括独特的分子标识符（UMIs），将读取计数折叠到其源 mRNA 计数，是否会提高福尔马林固定石蜡包埋（FFPE）肿瘤组织样本中转录本的定量。该检测（SET4）包括测量激素受体和 PI3-激酶相关转录活性（SET 和 PI3Kges）的特征，以及测量选定的激活点突变和关键乳腺癌基因的表达。
方法
修改包括在批量溶液中在逆转录（RT）期间引入八个核苷酸长的 UMIs 的步骤，随后在液滴中对标记的 cDNA 进行聚合酶链反应（PCR），并对聚合酶酶和反应条件进行优化。我们使用 Lin 的一致性相关系数（CCC）来衡量一致性，包括精度（Rho）和准确性（偏差），并使用非参数检验（Wilcoxon、Levene's）比较修改后的（NEW）SET4 检测与原始的（OLD）SET4 检测以及使用来自 12 个原发性乳腺癌的匹配新鲜冷冻（FF）和 FFPE 样本的全转录组 RNAseq。
结果
修改后的（NEW）SET4 检测在 FFPE 样本的技术重复中更准确地测量单个转录物（p<0.001）和 SET（p=0.002）。与 FF 样本相比，修改后的 SET4 检测在测量单个转录物方面更精确（Rho 0.966 与 0.888，p<0.01），但在测量多基因表达特征 SET（Rho 0.985 与 0.968）或 PI3Kges（Rho 0.985 与 0.946）方面则不然。与 FFPE 的 wtRNAseq 相比，它在测量转录物（Rho 0.986 与 0.934，p<0.001）和 SET（Rho 0.993 与 0.915，p=0.004）方面也更精确，但在测量 PI3Kges 方面则不然（Rho 0.988 与 0.945，p=0.051）。两种方案的准确性（偏差）相当。两个样本携带 PIK3CA 突变，并且在 FF 和 FFPE 样本中测量转录的突变等位基因分数相似，并且使用改良的 SET4 检测似乎更精确。当文库大小超过 400,000 个对齐读取时，FF 和 FFPE 样本中的扩增效率（每个 UMIs 的读取数）一致，并且接近理论预期值。
结论
对 SET4 检测的靶向 RNAseq 方案进行修改，显著提高了基于 UMIs 和基于读取的单个转录物、多基因特征和突变转录物分数的测量精度，特别是对 FFPE 样本。
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