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SMIXnorm: Fast and Accurate RNA-Seq Data Normalization for Formalin-Fixed Paraffin-Embedded Samples.

ABSTRACT: RNA-sequencing (RNA-seq) provides a comprehensive quantification of transcriptomic activities in biological samples. Formalin-Fixed Paraffin-Embedded (FFPE) samples are collected as part of routine clinical procedure, and are the most widely available biological sample format in medical research and patient care. Normalization is an essential step in RNA-seq data analysis. A number of normalization methods, though developed for RNA-seq data from fresh frozen (FF) samples, can be used with FFPE samples as well. The only extant normalization method specifically designed for FFPE RNA-seq data, MIXnorm, which has been shown to outperform the normalization methods, but at the cost of a complex mixture model and a high computational burden. It is therefore important to adapt MIXnorm for simplicity and computational efficiency while maintaining superior performance. Furthermore, it is critical to develop an integrated tool that performs commonly used normalization methods for both FF and FFPE RNA-seq data. We developed a new normalization method for FFPE RNA-seq data, named SMIXnorm, based on a simplified two-component mixture model compared to MIXnorm to facilitate computation. The expression levels of expressed genes are modeled by normal distributions without truncation, and those of non-expressed genes are modeled by zero-inflated Poisson distributions. The maximum likelihood estimates of the model parameters are obtained by a nested Expectation-Maximization algorithm with a less complicated latent variable structure, and closed-form updates are available within each iteration. Real data applications and simulation studies show that SMIXnorm greatly reduces computing time compared to MIXnorm, without sacrificing the performance. More importantly, we developed a web-based tool, RNA-seq Normalization (RSeqNorm), that offers a simple workflow to compute normalized RNA-seq data for both FFPE and FF samples. It includes SMIXnorm and MIXnorm for FFPE RNA-seq data, together with five commonly used normalization methods for FF RNA-seq data. Users can easily upload a raw RNA-seq count matrix and select one of the seven normalization methods to produce a downloadable normalized expression matrix for any downstream analysis. The R package is available at https://github.com/S-YIN/RSEQNORM. The web-based tool, RSeqNorm is available at http://lce.biohpc.swmed.edu/rseqnorm with no restriction to use or redistribute.

SUBMITTER: Yin S

PROVIDER: S-EPMC8024626 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundFormalin-fixed paraffin-embedded (FFPE) tissue samples are routinely archived in the course of patient care and can be linked to clinical outcomes with long-term follow-up. However, FFPE tissues have degraded RNA which poses challenges for analyzing gene expression. Next-generation sequencing (NGS) is rapidly becoming accepted as an effective tool for measuring gene expressions for research and clinical use. However, the feasibility of NGS has not been firmly established when using FFPE tissue.ResultsWe optimized strategies for whole transcriptome sequencing (RNA-seq) using FFPE tissue. Ribosomal RNA (rRNA) was successfully depleted by competitive hybridization using the Ribo-zero™ Kit (Epicentre Biotechnologies), and rRNA sequence content was less than one percent for each library. Gene expression measured by FFPE RNA-seq was compared to two different standards: RNA-seq from fresh frozen (FF) tissue and quantitative PCR (qPCR). Both FF and FFPE tumors were sequenced on an Illumina Genome Analyzer IIX with an average of 10 million reads. The distribution of FPKMs (fragments per kilobase of exon per million fragments mapped) and number of detected genes were similar between FFPE and FF. RNA-seq expressions from FF and FFPE samples from the same renal cell carcinoma (RCC) correlated highly (r = 0.919 for tumor 1 and r = 0.954 for tumor 2). On hierarchical cluster analysis, samples clustered by patient identity rather than method of preservation. TaqMan qPCR of 424 RCC-related genes correlated highly with FFPE RNA-seq expressions (r = 0.775 for FFPE tumor 1, r = 0.803 for FFPE tumor 2). Expression fold changes were considered, to assess biologic relevance of gene expressions. Expression fold changes between FFPE tumors (tumor 1/tumor 2) correlated well when comparing qPCR and RNA-seq (r = 0.890). Expression fold changes between tumors from different risk groups (our high risk RCC/The Cancer Genome Atlas, TCGA, low risk RCC) also correlated well when comparing RNA-seq from FF and FFPE tumors (r = 0.887).ConclusionsFFPE RNA-seq provides reliable genes expression data, comparable to that obtained from fresh frozen tissue. It represents a useful tool for discovery and validation of biomarkers.

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SMIXnorm: Fast and Accurate RNA-Seq Data Normalization for Formalin-Fixed Paraffin-Embedded Samples.

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