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Abstract：
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 (SETER/PR 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 SETER/PR (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 SETER/PR (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 SETER/PR (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.
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 SETER/PR (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 SETER/PR (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 SETER/PR (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.
摘要：
背景：我们的目标是评估对定制的靶向RNA测序（RNAseq）测定的修改是否包括将读段计数折叠到其源mRNA计数的独特分子标识符（UMI）将改善来自福尔马林固定的石蜡包埋（FFPE）肿瘤组织样品的转录物的定量。测定（SET4）包括测量激素受体和PI3激酶相关转录活性（SETER/PR和PI3Kges）的特征，并测量选定的激活点突变和关键乳腺癌基因的表达。
方法：修改包括在散装溶液中的逆转录（RT）过程中引入八个核苷酸长的UMI的步骤，然后对液滴中标记的cDNA进行聚合酶链反应（PCR），聚合酶和反应条件的优化。我们用林的一致性相关系数(CCC)来衡量一致性，包括精度(Rho)和准确度(偏差)，和非参数检验(Wilcoxon，Levene\s）使用来自12个原发性乳腺癌的匹配的新鲜冷冻（FF）和FFPE样品的RNA，将改进的（新）SET4测定与原始（旧）SET4测定以及整个转录组RNAseq进行比较。
结果：改进的（NEW）SET4测定法在FFPE样品的技术复制中更可重复地测量了单个转录本（p<0.001）和SETER/PR（p=0.002）。在FFPE中，改良的SET4测定法更精确地测量单转录本（Rho0.966vs0.888，p<0.01），但不测量多基因表达特征SETER/PR（Rho0.985vs0.968）或PI3Kges（Rho0.985vs0.946），与FF样品相比。它也比FFPE的wtRNAseq更精确地测量转录本（Rho0.986vs0.934，p<0.001）和SETER/PR（Rho0.993vs0.915，p=0.004），但不是PI3Kges(Rho0.988vs0.945，p=0.051)。方案之间的准确性（偏差）相当。两个样本携带PIK3CA突变,在FF和FFPE样品中，转录突变等位基因分数的测量结果相似，并且在改良的SET4测定中显得更加精确。扩增效率（每个UMI的读数）在FF和FFPE样品中是一致的，接近理论期望值，当库大小超过400,000个对齐读取时。
结论：对SET4测定的靶向RNAseq方案的修改显着提高了基于UMI和基于读取的单个转录本测量的精确度，多基因签名，和突变体转录分数，特别是FFPE样品。
方法：修改包括在散装溶液中的逆转录（RT）过程中引入八个核苷酸长的UMI的步骤，然后对液滴中标记的cDNA进行聚合酶链反应（PCR），聚合酶和反应条件的优化。我们用林的一致性相关系数(CCC)来衡量一致性，包括精度(Rho)和准确度(偏差)，和非参数检验(Wilcoxon，Levene\s）使用来自12个原发性乳腺癌的匹配的新鲜冷冻（FF）和FFPE样品的RNA，将改进的（新）SET4测定与原始（旧）SET4测定以及整个转录组RNAseq进行比较。
结果：改进的（NEW）SET4测定法在FFPE样品的技术复制中更可重复地测量了单个转录本（p<0.001）和SETER/PR（p=0.002）。在FFPE中，改良的SET4测定法更精确地测量单转录本（Rho0.966vs0.888，p<0.01），但不测量多基因表达特征SETER/PR（Rho0.985vs0.968）或PI3Kges（Rho0.985vs0.946），与FF样品相比。它也比FFPE的wtRNAseq更精确地测量转录本（Rho0.986vs0.934，p<0.001）和SETER/PR（Rho0.993vs0.915，p=0.004），但不是PI3Kges(Rho0.988vs0.945，p=0.051)。方案之间的准确性（偏差）相当。两个样本携带PIK3CA突变,在FF和FFPE样品中，转录突变等位基因分数的测量结果相似，并且在改良的SET4测定中显得更加精确。扩增效率（每个UMI的读数）在FF和FFPE样品中是一致的，接近理论期望值，当库大小超过400,000个对齐读取时。
结论：对SET4测定的靶向RNAseq方案的修改显着提高了基于UMI和基于读取的单个转录本测量的精确度，多基因签名，和突变体转录分数，特别是FFPE样品。
