Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Summary

This method describes the steps to improve the quality and quantity of sequence data that can be obtained from formalin-fixed paraffin-embedded (FFPE) RNA samples. We describe the methodology to more accurately assess the quality of FFPE-RNA samples, prepare sequencing libraries, and analyze the data from FFPE-RNA samples.

Abstract

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding of the basis of various diseases as well as resistance and/or susceptibility mechanisms. However, FFPE tissues, which represent the most common method for preserving tissue morphology in clinical specimens, are not the best sources for gene expression profiling analysis. The RNA obtained from such samples is often degraded, fragmented, and chemically modified, which leads to suboptimal sequencing libraries. In turn, these generate poor quality sequence data that may not be reliable for gene expression analysis and mutation discovery. In order to make the most of FFPE samples and obtain the best possible data from low quality samples, it is important to take certain precautions while planning experimental design, preparing sequencing libraries, and during data analysis. This includes the use of appropriate metrics for precise sample quality control (QC), identifying the best methods for various steps during the sequencing library generation, and careful library QC. In addition, applying correct software tools and parameters for sequence data analysis is critical in order to identify artifacts in RNA-seq data, filter out contamination and low quality reads, assess uniformity of gene coverage, and measure the reproducibility of gene expression profiles among biological replicates. These steps can ensure high accuracy and reproducibility for profiling of very heterogeneous RNA samples. Here we describe the various steps for sample QC, library preparation and QC, sequencing, and data analysis that can help to increase the amount of useful data obtained from low quality RNA, such as that obtained from FFPE-RNA tissues.

Introduction

Use of next-generation sequencing approaches has enabled us to glean a wealth of information from various types of samples. However, old and poorly preserved samples remain unworkable for the commonly used methods of generating sequence data and often require modifications to well-established protocols. FFPE tissues represent such a sample type that has been widely utilized for clinical specimens^1,2,3. While FFPE preservation maintains tissue morphology, the nucleic acids in FFPE tissues usually exhibit a wide range of damage and degradation, making it difficult to retrieve t....

Protocol

1. RNA quantity and quality assessment

1. Select the FFPE samples according to predefined criteria and extract RNA using an appropriate method (e.g., FFPE-nuclei acid extraction kit, Table of Materials).
   NOTE: There are several different methods available for FFPE-RNA extraction, including the newer microdissection methods that can work with very little tissue and extract good quality RNA^12,13,14.......

Discussion

The method described here outlines the main steps required to obtain good sequence data from FFPE-RNA samples. The main points to consider with this method are: (1) Ensure that the RNA is preserved as best as possible after extraction by minimizing the sample handling and freezing and thawing cycles. Separate QC aliquots are very helpful. (2) Use a QC metric that is best for the given sample set. RIN values and DV₂₀₀ are often not useful for degraded samples, and DV₁₀₀ may be the metric of choice to.......

Materials

Name	Company	Catalog Number	Comments
2100 Bioanalyzer	Agilent	G2939BA
Agilent DNA 7500 Kit	Agilent	5067-1506
Agilent High Sensitivity DNA Kit	Agilent	5067-4626
Agilent RNA 6000 Nano Kit	Agilent	5067-1511
AllPrep DNA/RNA FFPE Kit	Qiagen	80234
CFX96 Touch System	Bio-Rad	1855195
Library Quantification kit v2-Illumina	KapaBiosystems	KK4824
NEBNext Ultra II Directional RNA Library Prep Kit for Illumina	New England Biolabs	E7765S	https://www.neb.com/protocols/2017/02/07/protocol-for-use-with-ffpe-rna-nebnext-rrna-depletion-kit
NEBNext rRNA Depletion Kit (Human/Mouse/Rat)	New England Biolabs	E6310L
NextSeq 500 Sequencing System	Illumina	SY-415-1001	NextSeq 500 System guide: https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/nextseq/nextseq-500-system-guide-15046563-06.pdf
NextSeq PhiX Control Kit	Illumina	FC-110-3002
NSQ 500/550 Hi Output KT v2.5 (150 CYS)	Illumina	20024907
10X Genomics Magnetic Separator	10X Genomics	120250
Rotator Multimixer	VWR	13916-822
C1000 Touch Thermal Cycler	Bio-Rad	1851197
Sequencing reagent kit	Illumina	20024907
Flow cell package	Illumina	20024907
Buffer cartridge and the reagent cartridge	Illumina	20024907
Sodium hydroxide solution (0.2N)	Millipore Sigma	SX0607D-6
TRIS-HCL Buffer 1.0M, pH 7.0	Fisher Scientific	50-151-871