Jang Jin Sung, Holicky Eileen, Lau Julie, McDonough Samantha, Mutawe Mark, Koster Matthew J, Warrington Kenneth J, Cuninngham Julie M
Genome Analysis Core, Medical Genome Facility, Center for Individualized Medicine, Mayo Clinic, Stabile Research Building, 200 First Street SW, Rochester, MN, 55905, USA.
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.
BMC Genomics. 2021 Oct 24;22(1):759. doi: 10.1186/s12864-021-08068-1.
Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3' mRNA-seq method sequences the 3' region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3' mRNA-Seq using Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation.
The total mapped reads of QuantSeq 3' mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3' mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3' mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67).
In this study, QuantSeq 3' mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3' mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.
带有临床和组织学数据的存档福尔马林固定石蜡包埋（FFPE）组织样本是开发新分子生物标志物的极其宝贵的资源。然而，由于FFPE衍生的RNA样本常常高度修饰且片段化，转录组分析使用标准mRNA测序方法仍然具有挑战性。最近开发的3' mRNA测序方法使用独特分子标识符（UMI）对mRNA的3'区域进行测序，从而产生具有最小PCR偏差的基因表达数据。在本研究中，我们使用带有UMI的Lexogen QuantSeq 3' mRNA测序文库制备试剂盒FWD评估了3' mRNA测序的性能，并与TruSeq链特异性mRNA测序和RNA外显子捕获试剂盒进行比较。新鲜冷冻（FF）和FFPE组织产生的核苷酸大小范围为DV200值的13%至>70%；验证时的输入量范围为1 ng至100 ng。
在所有样本中，QuantSeq 3' mRNA测序与参考基因组的总比对读数范围为99%至74%。经过PCR偏差校正后，保留了3%至56%的总测序读数。QuantSeq 3' mRNA测序数据在1 ng至100 ng的输入量下，在通用人类参考RNA（UHR，R>0.94）的重复样本中显示出高度可重复的数据，在10 ng时，FF和FFPE配对样本（R = 0.92）也显示出高度可重复的数据。DV200值≤30%的严重降解的FFPE RNA在100 ng输入量时显示出良好的一致性（R>0.87）。直接将QuantSeq 3' mRNA测序数据与TruSeq链特异性mRNA测序（R = 0.78）和RNA外显子捕获数据（R>0.67）进行比较时，观察到中等相关性。
在本研究中，使用UMI进行PCR偏差校正的QuantSeq 3' mRNA测序被证明是一种适用于FF和FFPE RNA基因定量的方法。带有UMI的3' mRNA测序可应用于来自FFPE组织的严重降解的RNA，产生高质量的测序数据。
