We are thankful to Dr. Danielle Carrick (Division of Cancer Control and Population Sciences, National Cancer Institute) for continued help, especially for initiating this study, providing us with the samples, and for helpful suggestions during data analysis. We sincerely thank all members of the CCR Sequencing Facility at the Frederick National Laboratory for Cancer Research for their help during sample preparation and sequencing, especially Brenda Ho for assistance in sample QC, Oksana German for library QC, Tatyana Smirnova for running the sequencers. We also would like to thank Tsai-wei Shen and Ashley Walton at Sequencing Facility Bioinformatics Group for helping with data analysis and RNA-seq pipeline implementation. We also thank CCBR and NCBR for assistance with RNaseq analysis pipeline and best practices development.

1. RNA quantity and quality assessment
<ol>
	<li>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 RNA12,13,14...

<ol>
	<li>Carrick, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robustness+of+Next+Generation+Sequencing+on+Older+Formalin-Fixed+Paraffin-Embedded+Tissue.">Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.</a> PLoS One. 10 (7), 0127353 (2015).</li><li>Hedegaard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+of+RNA+and+DNA+isolated+from+paired+fresh-frozen+and+formalin-fixed+paraffin-embedded+samples+of+human+cancer+and+normal+tissue.">Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue.</a> PLoS One. 9 (5), 98187 (2014).</li><li>Zhang, P., Lehmann, B. D., Shyr, Y., Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utilization+of+Formalin+Fixed-Paraffin-Embedded+Specimens+in+High+Throughput+Genomic+Studies.">The Utilization of Formalin Fixed-Paraffin-Embedded Specimens in High Throughput Genomic Studies.</a> International Journal of Genomics. 2017, 1926304 (2017).</li><li>Srinivasan, M., Sedmak, D., Jewell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fixatives+and+tissue+processing+on+the+content+and+integrity+of+nucleic+acids.">Effect of fixatives and tissue processing on the content and integrity of nucleic acids.</a> American Journal of Pathology. 161 (6), 1961-1971 (2002).</li><li>von Ahlfen, S., Missel, A., Bendrat, K., Schlumpberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+RNA+quality+from+FFPE+samples.">Determinants of RNA quality from FFPE samples.</a> PLoS One. 2 (12), 1261 (2007).</li><li>Esteve-Codina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+RNA-Seq+Results+from+Paired+Formalin-Fixed+Paraffin-Embedded+and+Fresh-Frozen+Glioblastoma+Tissue+Samples.">A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples.</a> PLoS One. 12 (1), 0170632 (2017).</li><li>Vukmirovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+validation+of+differentially+expressed+transcripts+by+RNA-sequencing+of+formalin-fixed,+paraffin-embedded+(FFPE)+lung+tissue+from+patients+with+Idiopathic+Pulmonary+Fibrosis.">Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.</a> BMC Pulmonary Medicine. 17 (1), 15 (2017).</li><li>Adiconis, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+RNA+sequencing+methods+for+degraded+or+low-input+samples.">Comparative analysis of RNA sequencing methods for degraded or low-input samples.</a> Nature Methods. 10 (7), 623-629 (2013).</li><li>Sinicropi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+transcriptome+RNA-Seq+analysis+of+breast+cancer+recurrence+risk+using+formalin-fixed+paraffin-embedded+tumor+tissue.">Whole transcriptome RNA-Seq analysis of breast cancer recurrence risk using formalin-fixed paraffin-embedded tumor tissue.</a> PLoS One. 7 (7), 40092 (2012).</li><li>Altekruse, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEER+cancer+registry+biospecimen+research:+yesterday+and+tomorrow.">SEER cancer registry biospecimen research: yesterday and tomorrow.</a> Cancer Epidemiology, Biomarkers &amp; Prevention. 23 (12), 2681-2687 (2014).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robustness+of+RNA+sequencing+on+older+formalin-fixed+paraffin-embedded+tissue+from+high-grade+ovarian+serous+adenocarcinomas.">Robustness of RNA sequencing on older formalin-fixed paraffin-embedded tissue from high-grade ovarian serous adenocarcinomas.</a> PLoS One. 14 (5), 0216050 (2019).</li><li>Amini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimised+protocol+for+isolation+of+RNA+from+small+sections+of+laser-capture+microdissected+FFPE+tissue+amenable+for+next-generation+sequencing.">An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.</a> BMC Molecular Biology. 18 (1), 22 (2017).</li><li>Amini, P., Nassiri, S., Ettlin, J., Malbon, A., Markkanen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+RNA+sequencing+of+FFPE+subsections+reveals+highly+conserved+stromal+reprogramming+between+canine+and+human+mammary+carcinoma.">Next-generation RNA sequencing of FFPE subsections reveals highly conserved stromal reprogramming between canine and human mammary carcinoma.</a> Disease Models and Mechanisms. 12 (8), (2019).</li><li>Wimmer, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evaluation+of+RNA+quality,+microarray+data+reliability+and+pathway+analysis+in+fresh,+fresh+frozen+and+formalin-fixed+paraffin-embedded+tissue+samples.">Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.</a> Scientific Reports. 8 (1), 6351 (2018).</li><li>. Babraham Bioinformatics Available from: <a target="_blank" href="https://www.bioinformatics.babraham.ac.uk/projects/fastqc/">https://www.bioinformatics.babraham.ac.uk/projects/fastqc/</a> (2019)</li><li>Martin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutadapt+removes+adapter+sequences+from+high-throughput+sequencing+reads.">Cutadapt removes adapter sequences from high-throughput sequencing reads.</a> EMBnet.journal. 17 (1), 10-12 (2011).</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>. Babraham Bioinformatics Available from: <a target="_blank" href="https://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/">https://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/</a> (2019)</li><li>Wood, D. E., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kraken:+ultrafast+metagenomic+sequence+classification+using+exact+alignments.">Kraken: ultrafast metagenomic sequence classification using exact alignments.</a> Genome Biology. 15 (3), 46 (2014).</li><li>Dobin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAR:+ultrafast+universal+RNA-seq+aligner.">STAR: ultrafast universal RNA-seq aligner.</a> Bioinformatics. 29 (1), 15-21 (2013).</li><li>Wang, L., Wang, S., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSeQC:+quality+control+of+RNA-seq+experiments.">RSeQC: quality control of RNA-seq experiments.</a> Bioinformatics. 28 (16), 2184-2185 (2012).</li><li>Ewels, P., Magnusson, M., Lundin, S., Kaller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MultiQC:+summarize+analysis+results+for+multiple+tools+and+samples+in+a+single+report.">MultiQC: summarize analysis results for multiple tools and samples in a single report.</a> Bioinformatics. 32 (19), 3047-3048 (2016).</li><li>Li, B., Dewey, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSEM:+accurate+transcript+quantification+from+RNA-Seq+data+with+or+without+a+reference+genome.">RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.</a> BMC Bioinformatics. 12, 323 (2011).</li><li>Son, K., Yu, S., Shin, W., Han, K., Kang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Guideline+to+Assess+the+Characteristics+of+RNA-Seq+Data.">A Simple Guideline to Assess the Characteristics of RNA-Seq Data.</a> BioMed Research International. 2018, 2906292 (2018).</li><li>McCarthy, D. J., Chen, Y., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+analysis+of+multifactor+RNA-Seq+experiments+with+respect+to+biological+variation.">Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.</a> Nucleic Acids Research. 40 (10), 4288-4297 (2012).</li><li>Robinson, M. D., McCarthy, D. J., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edgeR:+a+Bioconductor+package+for+differential+expression+analysis+of+digital+gene+expression+data.">edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.</a> Bioinformatics. 26 (1), 139-140 (2010).</li><li>Ritchie, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=limma+powers+differential+expression+analyses+for+RNA-sequencing+and+microarray+studies.">limma powers differential expression analyses for RNA-sequencing and microarray studies.</a> Nucleic Acids Research. 43 (7), 47 (2015).</li><li>Subramanian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+set+enrichment+analysis:+a+knowledge-based+approach+for+interpreting+genome-wide+expression+profiles.">Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.</a> Proceedings of the National Academy of Sciences of the United States of America U S A. 102 (43), 15545-15550 (2005).</li><li>Mootha, V. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGC-1alpha-responsive+genes+involved+in+oxidative+phosphorylation+are+coordinately+downregulated+in+human+diabetes.">PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes.</a> Nature Genetics. 34 (3), 267-273 (2003).</li><li>Ashburner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+ontology:+tool+for+the+unification+of+biology.+The+Gene+Ontology+Consortium.">Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.</a> Nature Genetics. 25 (1), 25-29 (2000).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nature Protocols. 4 (1), 44-57 (2009).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatics+enrichment+tools:+paths+toward+the+comprehensive+functional+analysis+of+large+gene+lists.">Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.</a> Nucleic Acids Research. 37 (1), 1-13 (2009).</li><li>Evaluating RNA Quality from FFPE Samples. Illumina Available from: <a target="_blank" href="https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf">https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf</a> (2016)</li></ol>

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...

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 specimens1,2,3. 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 the genomic information that may lead to important insights about molecular mechanisms underlying various disorders.
Gene expression data generated by RNA sequencing is often instrumental in studying disease and resistance mechanisms and complements DNA mutation analysis. However, RNA is more susceptible to degradation, making it more challenging to generate accurate gene expression data from FFPE tissues. Furthermore, because the wide availability and affordability of sequencing is relatively recent, older specimens were often not stored in conditions required to preserve RNA integrity. Some of the issues for FFPE samples include degradation of RNA due to embedding in paraffin, chemical modification of RNA leading to fragmentation or refractoriness to enzymatic processes required for sequencing, and loss of the poly-A tails, limiting the applicability of oligo-dT as a primer for reverse transcriptase4. Another challenge is the handling/storage of FFPE samples under suboptimal conditions, which may lead to further degradation of labile molecules such as RNA in the tissues5. This is especially relevant for older samples that may have been collected at a time when gene expression analysis by RNA sequencing was not anticipated for the samples. All these lead to decreased quality and quantity of the extracted RNA available for generating useful sequence data. The low probability of success, combined with the high cost of sequencing, has dissuaded many researchers from trying to generate and analyze gene expression data from potentially useful FFPE samples. Some studies in recent years have demonstrated the usability of FFPE tissues for gene expression analysis2,6,7,8,9, albeit for fewer and/or more recent samples.
As a feasibility study, we used RNA extracted from FFPE tumor tissue specimens from three Residual Tissue Repositories from Surveillance, Epidemiology, and End Results (SEER) cancer registries for RNA sequencing and gene expression analysis10. Procured from clinical pathology labs, the FFPE tissues from high-grade ovarian serous adenocarcinomas were stored from 7&#8211;32 years under varying conditions before RNA extraction. Because in most cases these blocks had been stored in different sites for years without the expectation of any sensitive genetic analysis in the future, not much care had been taken to preserve the nucleic acids. Thus, most of the samples exhibited poor quality RNA, with a large proportion of samples contaminated with bacteria. Nevertheless, we were able to perform gene quantification, measure the uniformity and continuity of gene coverage, and perform the Pearson correlation analysis among biological replicates to measure reproducibility. Based on a set of key signature gene panel, we compared the samples in our study with The Cancer Genome Atlas (TCGA) data and confirmed that approximately 60% of the samples had comparable gene expression profiles11. Based on the correlation between various QC results and sample metadata, we identified key QC metrics that have good predictive value for identifying samples that are more likely to generate usable sequence data11.
Here we describe the methodology used for FFPE-RNA quality assessment, generation of sequencing libraries starting from extracted RNA samples, and bioinformatic analysis of the sequencing data.

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

optimization for sequencing and analysis of degraded ffpe-rna samples

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.

The methodology described above was applied to 67 FFPE samples that had been stored under a variety of different conditions for 7&#8211;32 years (the median sample storage time was 17.5 years). The dataset and analysis results presented here were previously described and published in Zhao et al.11. On checking the sample quality as described earlier (i.e., example traces in Figure 2), DV100 was found to be more useful than DV200 because it is mor...

Watch this Scientific Journal Video about Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples at JoVE.com

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

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.

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding ...

Our protocol can be used to improve the quality and quantity of gene expression data generated from FFPE-derived RNA samples, especially in suboptimal quality samples. This technique assesses the RNA quality within FFPE tissue samples and enables optimization of their downstream processing to improve the quantity and quality of the sample sequencing data. This method allow the comprehensive characterization of a transcript in biological and clinical sample and may provide insight into functional elements of the genome in development and disease.The FFPE-RNA degradation and selection of the methods for sample QC, library preparation, and data analysis can all be very tricky. Be meticulous when selecting the right methods and appropriate controls. Visual demonstration allows viewers to observe the small subtle modifications and precautions needed during the planning, assessment, and the sequencing process especially for poorly preserved FFPE samples.To check the quality of the RNA sample, run the sample on an RNA QC system according to the manufacturer's instructions and use the appropriate bioanalysis software to analyze the distribution of the RNA fragments within the sample and to calculate the proportion of fragments longer than 100 and 200 nucleotides. Based on the QC metrics, identify the samples with fewer than 40%of the fragments longer than 100 nucleotides to allow exclusion of these samples from processing. For sequencing, thaw the appropriate sequencing reagent kits according to the run parameters in a room temperature water bath and place the reagent tray at four degrees Celsius after thawing.Place the flow cell package at room temperature for 30 minutes. While the package is warming, open the Illumina Experiment Manager application and select Create Sample Sheet. Select the sequencer to be used and click Next.Enter the reagent kit barcode and the other appropriate workflow parameters. Then upload a sample sheet created according to the Illumina sequence criteria. To prepare the sample library and the control library PhiX, denature and dilute the libraries to the appropriate concentrations and mix the sample and PhiX control libraries to obtain a 5%PhiX control volume ratio.When the flow cells are ready, remove the wrapping, clean the glass surface with a lint-free alcohol wipe, and dry the glass with a low lint laboratory tissue. Remove the reagent cartridge from four degrees Celsius storage and invert the cartridge five times to mix the reagents. Gently tap the cartridge on the bench to reduce air bubbles and load the denatured and diluted sample into the reagent cartridge in the designated reservoir.Load the flow cell, buffer cartridge, and the reagent cartridge onto the system. Then perform an automated check and review the output to ensure that the run parameters pass the system check. When the automated check is complete, select start to begin the sequencing run.To visualize the pre-processing QC and post-alignment QC results, run multi-QC to generate an aggregated report in HTML format. To determine batch effects and to assess the quality map of the given dataset, use an R script to run a principal component analysis. Sample correlation analysis can then be carried out using the Pearson correlation between different samples.The estimation of the proportion of fragments longer than 100 nucleotides is more useful than an estimation of the fragments longer than 200 nucleotides because it is more sensitive for accurately measuring the proportion of smaller fragment sizes for highly degraded RNA samples. Given the high degree of degradation in the sample set, a total RNA library preparation method is recommended. For example, here sequencing libraries prepared from four different samples are shown.Here, overviews of raw FastQ file sequencing quality and sample adapter content are shown. FastQC screening can help detect contamination such as bacterial or mouse contamination within the samples. STAR alignment can be used to determine the proportion of reads mapped to the reference genome, the percentage of reads uniquely mapped to the reference genome, and the proportion of reads that were not mapped, or that were mapped to multiple loci.The card and RSeQC statistics can be used to determine the percent of messenger RNAs, intronic and intergenic bases present in the mapped reads. For these representative analyses, some degraded libraries had a three prime bias in which more reads were mapped closer to the three prime than to the five prime end. In addition, principal component analysis can be performed to determine the percent of the total variation captured by the principal components for the biological replicates of each sample.Take care to avoid sample degradation, prepare replicates and multiple coats for each sample, use the appropriate metrics to assess the sample quality, and use the right library preparation method. Using this methodology, larger NGS studies can be performed with previously unusable FFPE samples with varying storage times and conditions to gain insights into a variety of different disease states. This technique paved the way for researchers to study existing large archives of FFPE sample with rich clinical information and can greatly enhance population-based cancer studies.

optimization-for-sequencing-and-analysis-of-degraded-ffpe-rna-samples

Research

JoVE Journal

Genetics

11.7K 次观看.  Frederick National Laboratory for Cancer Research. 我们的协议可用于提高从FFPE衍生的RNA样本生成的基因表达数据的质量和数量，特别是在质量欠佳的样品中。该技术评估FFPE组织样品中的RNA质量，并优化其下游处理，以提高样品测序数据的数量和质量。此方法允许在生物和临床样本中全面鉴定成绩单，并可能提供对基因组在发育和疾病中的功能元素的洞察。FFPE-RNA降解和样品质量控制方法的选择、库制备和数据分析都十...