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.</li>
	<li>Utmost care should be taken to preserve the integrity of RNA at all stages. This includes working with RNase free deionised water, using RNase free plasticware, and cleaning all instruments that come in contact with the FFPE blocks with RNase decontamination reagents.</li>
	<li>RNA should always be handled carefully and kept in ice unless otherwise specified to minimize degradation while handling.</li>
	<li>If enough material is available, extract RNA from more than one region in the FFPE block to generate biological replicates from as many samples as possible. For some of the samples with ample RNA yield, divide the extracted RNA into two to process as technical replicates.</li>
	<li>If possible, collect a small amount of sample separately after extraction for QC (i.e., a QC aliquot) to avoid repeated handling and freeze-thaw cycles of the sample that will likely lead to degradation of the RNA.</li>
	<li>Check the quality of the RNA (preferably from the QC aliquot) by running it on an RNA QC system (e.g., Agilent Bioanalyzer system using an RNA Nano chip, Table of Materials) according to the manufacturer&#8217;s instructions.</li>
	<li>Analyze the distribution of RNA fragments in the samples (e.g., using the Bioanalyzer 2100 Expert software) by calculating the DV200 and DV100 values as the percent of fragments larger than 200 nt (DV200) or 100 nt (DV100) in size.</li>
	<li>Among DV200 and DV100, identify the metric that has a larger spread of values for the given sample set, and pick that for grouping the samples according to their degree of intactness. 
	NOTE: For sample sets with more intact RNA molecules (i.e., high DV200 values, all or most with DV200 &#62; 40%), DV200 is likely to be a useful QC metric. However, for sample sets with more degraded transcripts (i.e., low DV200 values, all or most with DV200 &#60; 40%), DV100 is more likely to be useful.</li>
	<li>Based on the QC metrics, identify the samples that have DV100 &#60; 40%. Because this degree of degradation is highly likely to not generate useful sequencing data11, it is advisable to avoid processing such samples. If replacements for such samples are available, their quality should be checked to ideally only include samples with DV100 &#62; 50%.</li>
</ol>
2. Sequencing library preparation
<ol>
	<li>Based on the quality of the samples as assessed in section 1, identify an appropriate method for generating the sequencing libraries.
	<ol>
		<li>For sample sets with very low degradation and high DV200 values, use mRNA sequencing (i.e., capture of polyadenylated transcripts), targeted RNA sequencing (i.e., use of capture probes for specific genes of interest), RNA exome sequencing (i.e., use of capture probes to enrich for the coding transcriptome), or total RNA sequencing (i.e., use of random primers for reverse transcription to sequence the entire RNA population after removing ribosomal RNA from the samples). However, it is important to note that the fixation process may introduce bias in the extracted RNA. Thus, the capture approaches may not work well in all cases, even with high DV200 values.</li>
		<li>If the sample set includes samples with high degradation (DV200 &#60; 30%), use a total RNA library preparation method and not one that depends on the capture of specific regions of the transcripts, because those specific regions may be missing in degraded samples. The use of random primers for generation of cDNA leads to higher representation of usable RNA in the final library, and is, therefore, more suited for FFPE-RNA samples.</li>
		<li>For ribosomal RNA depletion for sample sets with high degradation, use RNaseH-based methods. These are methods where rRNA-specific DNA probes bind to rRNA, double-stranded molecules are digested by RNaseH, and leftover probes are cleaned up by DNase (e.g., NEBNext rRNA depletion kit, Table of Materials). These methods work better for degraded samples than some other methods8.</li>
	</ol>
	</li>
	<li>For generating sequencing libraries, use higher input amounts (if possible) for samples that have more degraded RNA (DV100 &#60; 60%). While samples with reasonably good quality RNA (DV100 &#62; 60%) may yield good sequence data even at lower input amounts (the lowest tested for this protocol with FFPE-RNA was ~20 ng), for more degraded RNA (DV100 &#60; 60%), it is better to start with higher input amounts (e.g., &#62;100 ng). 
	NOTE: If enough (e.g., &#62;500 ng) sample is available, it is advisable to save at least half of the sample for repeating the library preparation, if needed. For low input samples (e.g., &#60;100 ng), it is usually better to use the entire amount and generate a library of sufficient diversity.</li>
	<li>After selecting a suitable library preparation kit for generating total RNA seq libraries from samples with high degradation (e.g., NEBNext Ultra II RNA Library Prep Kit for Illumina, see Table of Materials), follow the manufacturer&#8217;s instructions to generate the libraries. 
	NOTE: During library preparation, it is important to skip the RNA fragmentation step for degraded samples and to ensure the use of random primers for first strand cDNA synthesis.</li>
	<li>For improving the efficiency and speed, especially for the low-input samples, use appropriate magnetic racks with strong fixed magnets for bead-based purification and size-selection steps (see Table of Materials).</li>
	<li>For PCR enrichment of adapter ligated DNA, adjust the number of amplification cycles based on the amount of input DNA to ensure maximum representation while avoiding unnecessary duplication of the library molecules. For low input FFPE-RNA samples (&#60;100 ng), we recommend 16&#8211;18 amplification cycles, while the high input samples (1,000 ng) usually generate enough library amounts in 12&#8211;14 rounds of amplification.</li>
	<li>Following PCR amplification and cleanup per the manufacturer&#8217;s instructions, assess the library quality by analyzing library concentration and molecule distribution on an appropriate platform (e.g., Agilent Bioanalyzer DNA Chip, see Table of Materials). For samples with primer peaks (~80 bp) or adapter-dimer peaks (~128 bp), repeat the cleanup to remove those peaks.</li>
	<li>Calculate the average library size for each library (e.g., using the Bioanalyzer 2100 Expert software).</li>
</ol>
3. Sequencing library QC
<ol>
	<li>Once it has been ascertained that the libraries are free of excess primer and adapter-dimers and have sufficient concentration for subsequent sequencing, quantitate further by qPCR. 
	NOTE: Owing to the sensitivity of cluster generation towards library concentration, accurate quantification is vital to prevent costly sequencing runs from underperformance or overloading. Quantitative real-time PCR (qPCR) methods are useful for improving cluster density on Illumina platforms without resulting in overclustering. The qPCR method is more precise and more sensitive than the methods based on qualitative and/or quantitative analysis of all library molecules (e.g., Agilent Bioanalyzer), because it measures the templates that have both adapter sequences on either end that will form clusters on the flowcell. Library size must, however, be known in advance as a size correction must be applied to all samples so that results can be compared against a standard curve. 
	CAUTION: Lab coats and gloves must always be worn when performing qPCR, and the procedure must be performed in a biosafety cabinet following the manufacturer&#8217;s instructions.
	<ol>
		<li>Set up a 96 well plate with three replicates for each sample for error prevention using a suitable kit (e.g., KAPA SYBR FAST qPCR Master Mix for Illumina libraries, a part of Library Quantification kit, see Table of Materials), along with the standards, a positive control (e.g, PhiX control, see Table of Materials), and a no template control (NTC). The NTC is qPCR mix without DNA library. The positive control can be any library with known concentration and fragment size.
		<ol>
			<li>Prepare a minimum of six dilutions of the standards following the vendor protocol.</li>
		</ol>
		</li>
		<li>After adding all the components (i.e., qPCR master mix, libraries, standards), cover the plate with sealing film and use a squeegee to ensure the film makes even and secure contact with the plate.</li>
		<li>Vortex and spin down the plate at 1,500 rpm for at least 1 min. Visually inspect the plate to make sure there are no air bubbles at the bottom of the wells.</li>
		<li>Set up the plate on the thermal cycler (e .g. CFX96 Touch System, see Table of Materials) using the manufacturer&#39;s recommended settings.</li>
		<li>Save the run folder where it can be accessed for data analysis.</li>
		<li>During data analysis, check that the slope is in the -3.1 to -3.6 range, efficiency from 90% to 110% and the R2 (coefficient of correlation obtained for the standard curve) no less than 0.98.</li>
	</ol>
	</li>
	<li>Pooling: Once the qPCR concentration of the sequencing ready libraries is obtained, pool equimolar amounts of each of the libraries, depending on the number of sequencing reads required per sample and the sequencing output of the instrument.</li>
	<li>QC of the pools: Quantitate the library pools again by qPCR following the same protocol as described in step 3.1.</li>
</ol>
4. Sequencing
<ol>
	<li>Depending on the run parameters, pull the sequencing reagent kits and thaw them following the user guide. Please check the Illumina website for the latest versions of all user guides for sequencing on Illumina instruments.</li>
	<li>Make sure the reagents are completely thawed and place the reagents tray at 4 &#176;C. The run should be started no later than 2 h after the reagents have been defrosted. Not doing that could affect quality of the run results.</li>
	<li>Invert the cartridge 5x to mix reagents and gently tap on the bench to reduce air bubbles.</li>
	<li>Set the unwrapped flow cell package aside at room temperature for 30 min.</li>
	<li>Unwrap the flow cell package and clean the glass surface of the flow cell with a lint-free alcohol wipe. Dry the glass with a low-lint laboratory tissue.</li>
	<li>Open the Illumina &#8220;Experiment Manager&#8221; application. Choose &#8220;Create Sample Sheet&#8221;, then choose the Sequencer and click &#8220;Next&#8221;.</li>
	<li>Create and upload the sample sheet based on Illumina sequencer criteria (e.g., Illumina Experiment Manager, software guide).</li>
	<li>At the prompts, scan in the reagent kit barcode and enter the run Set Up Parameters (e.g., for a single indexed PE 75 cycle run, enter 76-8-76).</li>
	<li>Denature and dilute the library pool based on the sequencer user guide recommendation (e.g., NextSeq 500 System guide from Illumina, see Table of Materials).</li>
	<li>Denature and dilute the control library PhiX (see Table of Materials) to the appropriate concentration (e.g., 1.8 pM for NextSeq).</li>
	<li>Mix sample library and PhiX control to result in a 1% PhiX control volume ratio.</li>
	<li>Load denatured and diluted sample into the reagent cartridge in the designated reservoir.</li>
	<li>Load the flowcell, buffer cartridge, and the reagent cartridge.</li>
	<li>Perform an automated check and review to ensure that the run parameters pass the system check.</li>
	<li>When the automated check is complete, select Start to begin the sequencing run.</li>
</ol>
5. Data analysis and quality assessment
NOTE: A typical RNA-seq data analysis workflow (Figure 1) includes preprocessing and QC, alignment to genome and post alignment QC, gene and transcript quantification, sample correlation analysis, differential analysis between different sample groups, treatment conditions, and gene set enrichment and pathway analysis.
The RNA-seq data may have quality issues that can affect the accuracy of gene profiling and lead to erroneous conclusions. Therefore, initial QC checks for sequencing quality, contamination, sequencing coverage bias, and other sources of artifacts are very important. Applying an RNA-Seq QC pipeline similar to the workflow described here is recommended to detect artifacts and apply filtering or correction before downstream analysis.
<ol>
	<li>Preprocessing 
	NOTE: This includes demultiplexing, assessment of sequence read quality, GC content, presence of sequencing adapters, overrepresented k-mers, and PCR duplicated reads. This information helps to detect sequencing errors, PCR artifacts, or contamination.
	<ol>
		<li>Demultiplex Illumina sequencing run using the Illumina software tool bcl2fastq2 to generate raw FASTQ files for each sample defined in the sample sheet. Allow one mismatch in the sample index barcodes to tolerate sequencing errors if there is no barcode collision.</li>
		<li>Run the FASTQC15 software tool to perform a quality check on raw FASTQ files to detect any poor quality or abnormalities in sequencing reads.</li>
		<li>For adapter and low-quality bases trimming, trim the sequencing adapters and low quality bases using Cutadapt16 or Trimmomatic17 software tools. Save the trimmed reads in the pair-end fastq files.</li>
		<li>Contamination screen
		<ol>
			<li>Run FASTQ_screen18 to detect possible cross contamination with other species.</li>
			<li>Run miniKraken of Kraken219 to identify the taxonomies of contaminating species.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Alignment to reference genome and post alignment QC
	<ol>
		<li>The trimmed reads can be aligned to a reference genome sequence (GRCh Build hg19 or hg38) using STAR aligner20. Apply the Gencode annotation GTF file to guide the spliced transcript alignment. It is recommended to run STAR 2-pass to increase sensitivity to novel splice junctions. In the second pass, all reads will be remapped using annotated gene and transcripts and novel junctions from the first pass.</li>
		<li>Perform post-alignment QC.
		<ol>
			<li>Run Picard&#8217;s21 MarkDuplicates to evaluate the library complexity by determining the amount of unique or nonduplicated reads in the samples.</li>
			<li>Run Picard&#8217;s CollectRnaSeqMetrics program to collect mapping percentages on coding, intronic, intergenic, UTR regions, and gene body coverage.</li>
			<li>Run RSeQC22 to determine the read pair inner distance, read distribution among CDS exons, 5&#8217;UTR, 3&#8217;UTR, intron, TSS_up_1kb, TSS_up_5kb, TSS_up_10kb, TES_down_1kb, TES_down_5kb, TES_down_10kb, read GC content, junction saturation, and library strand information.</li>
			<li>Run multi-QC23 to generate an aggregated report in HTML format.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Gene quantification and correction analysis
	<ol>
		<li>Run RSEM24 to get raw count as well as normalized read count on genes and transcripts. The read count measurement such as RPKM (reads per kilobase of exon model per million reads), FPKM (fragments per kilobase of exon model per million mapped reads), and TPM (transcripts per million) are the most often reported RNA-seq gene expression values. Genes expressed below a noised threshold (such as TPM &#60; 1 or raw count &#60;5) can be filtered.</li>
		<li>Perform transcript quantification to aggregate raw counts of mapped reads to each transcript sequences using programs such as HTSeq-count or featureCounts.</li>
		<li>Run Principal Components Analysis (PCA) using an R script to determine batch effects and assess a quality map of the given dataset25. Sample correlation analysis can be carried out using the Pearson correlation between different metrics.</li>
	</ol>
	</li>
	<li>Differential gene expression analysis
	<ol>
		<li>Perform gene differential analysis between sample conditions using the program edgeR26,27 and/or limma-Voom28 and use normalization methods including TPM, TMM, DESeq, or UpperQuartile.</li>
		<li>It is recommended to run at least two differential analysis software tools in order to call two set of DEGs lists for comparison and get the final DEGs to improve detection sensitivity and accuracy.</li>
	</ol>
	</li>
	<li>Gene set enrichment and pathway analysis
	<ol>
		<li>Perform Gene Set Enrichment Analysis (GSEA)29,30 based on ranking of transcripts according to a measurement of differentially expressed genes (DEGs) list to determine if the DEGs show statistically significant, concordant differences between biological conditions.</li>
		<li>Perform function analysis using resources such as Gene Ontology31, DAVID32,33, or other available software tools.</li>
	</ol>
	</li>
</ol>

<ol><li>Carrick, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robustness+of+Next+Generation+Sequencing+on+Older+Formalin-Fixed+Paraffin-Embedded+Tissue%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((The+Utilization+of+Formalin+Fixed-Paraffin-Embedded+Specimens+in+High+Throughput+Genomic+Studies%5BTitle%5D)+AND+%22International+Journal+of+Genomics%22%5BJournal%5D)">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/pubmed?term=((Effect+of+fixatives+and+tissue+processing+on+the+content+and+integrity+of+nucleic+acids%5BTitle%5D)+AND+%22American+Journal+of+Pathology%22%5BJournal%5D)">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/pubmed?term=((Determinants+of+RNA+quality+from+FFPE+samples%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((A+Comparison+of+RNA-Seq+Results+from+Paired+Formalin-Fixed+Paraffin-Embedded+and+Fresh-Frozen+Glioblastoma+Tissue+Samples%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Identification+and+validation+of+differentially+expressed+transcripts+by+RNA-sequencing+of+formalin-fixed%2C+paraffin-embedded+%28FFPE%29+lung+tissue+from+patients+with+Idiopathic+Pulmonary+Fibrosis%5BTitle%5D)+AND+%22BMC+Pulmonary+Medicine%22%5BJournal%5D)">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/pubmed?term=((Comparative+analysis+of+RNA+sequencing+methods+for+degraded+or+low-input+samples%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">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/pubmed?term=((Whole+transcriptome+RNA-Seq+analysis+of+breast+cancer+recurrence+risk+using+formalin-fixed+paraffin-embedded+tumor+tissue%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((SEER+cancer+registry+biospecimen+research%3A+yesterday+and+tomorrow%5BTitle%5D)+AND+%22Cancer+Epidemiology%2C+Biomarkers+%26+Prevention%22%5BJournal%5D)">SEER cancer registry biospecimen research: yesterday and tomorrow.</a> Cancer Epidemiology, Biomarkers & Prevention. 23 (12), 2681-2687 (2014).</li>
<li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robustness+of+RNA+sequencing+on+older+formalin-fixed+paraffin-embedded+tissue+from+high-grade+ovarian+serous+adenocarcinomas%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((An+optimised+protocol+for+isolation+of+RNA+from+small+sections+of+laser-capture+microdissected+FFPE+tissue+amenable+for+next-generation+sequencing%5BTitle%5D)+AND+%22BMC+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Next-generation+RNA+sequencing+of+FFPE+subsections+reveals+highly+conserved+stromal+reprogramming+between+canine+and+human+mammary+carcinoma%5BTitle%5D)+AND+%22Disease+Models+and+Mechanisms%22%5BJournal%5D)">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/pubmed?term=((Systematic+evaluation+of+RNA+quality%2C+microarray+data+reliability+and+pathway+analysis+in+fresh%2C+fresh+frozen+and+formalin-fixed+paraffin-embedded+tissue+samples%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Cutadapt+removes+adapter+sequences+from+high-throughput+sequencing+reads%5BTitle%5D)+AND+%22EMBnet.journal%22%5BJournal%5D)">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/pubmed?term=((Trimmomatic%3A+a+flexible+trimmer+for+Illumina+sequence+data%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((Kraken%3A+ultrafast+metagenomic+sequence+classification+using+exact+alignments%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">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/pubmed?term=((STAR%3A+ultrafast+universal+RNA-seq+aligner%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">STAR: ultrafast universal RNA-seq aligner.</a> Bioinformatics. 29 (1), 15-21 (2013).</li>
<li>Broad Institute. , Available from: 
 <a target="_blank" href="http://broadinstitute.github.io/picard/">http://broadinstitute.github.io/picard/</a> (2019).</li>
<li>Wang, L., Wang, S., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RSeQC%3A+quality+control+of+RNA-seq+experiments%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((MultiQC%3A+summarize+analysis+results+for+multiple+tools+and+samples+in+a+single+report%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((RSEM%3A+accurate+transcript+quantification+from+RNA-Seq+data+with+or+without+a+reference+genome%5BTitle%5D)+AND+%22BMC+Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((A+Simple+Guideline+to+Assess+the+Characteristics+of+RNA-Seq+Data%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">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/pubmed?term=((Differential+expression+analysis+of+multifactor+RNA-Seq+experiments+with+respect+to+biological+variation%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">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/pubmed?term=((edgeR%3A+a+Bioconductor+package+for+differential+expression+analysis+of+digital+gene+expression+data%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((limma+powers+differential+expression+analyses+for+RNA-sequencing+and+microarray+studies%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">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/pubmed?term=((Gene+set+enrichment+analysis%3A+a+knowledge-based+approach+for+interpreting+genome-wide+expression+profiles%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America+U+S+A%22%5BJournal%5D)">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/pubmed?term=((PGC-1alpha-responsive+genes+involved+in+oxidative+phosphorylation+are+coordinately+downregulated+in+human+diabetes%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">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/pubmed?term=((Gene+ontology%3A+tool+for+the+unification+of+biology.+The+Gene+Ontology+Consortium%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">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/pubmed?term=((Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Bioinformatics+enrichment+tools%3A+paths+toward+the+comprehensive+functional+analysis+of+large+gene+lists%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">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>

This work was funded by the National Cancer Institute (NCI), National Institutes of Health (NIH). Leidos Biomedical Research, Inc. is the operations and technical support contractor for the Frederick National Laboratory for Cancer Research which is fully funded by NIH. Several authors (YZ, MM, KT, YL, JS, BT) are affiliated with Leidos Biomedical Research, Inc., but all of the authors are fully funded by the National Cancer Institute including authors&#8217; salaries and research materials. Leidos Biomedical Research, Inc. did not provide salary for the authors (YZ, MM, KT, YL, JS, BT) or material for the study, nor did it have any role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.

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 assess the quality in a given sample set. (3) For more degraded samples, it is best to use a high sample input. Higher input amounts lead to better diversity and lower duplication in the final library, leading to improved data quality. Because not all RNA in FFPE-RNA samples is usable due to high degradation and refractoriness to enzymatic processes, these effects are more pronounced in FFPE-RNA compared to fresh frozen RNA. (4) Use random priming for the reverse transcription step as opposed to the use of oligo-dT or specific sequences as primers. Unless the set of specific probes is able to cover as much sequence as possible for all transcripts of interest, random primers are a safe bet to ensure the conversion of a maximum number of transcripts (or fragments thereof) into cDNA. Thus, total RNA library prep methods are more useful for degraded samples than mRNA methods, which rely on the presence of poly-A tails. (5) Accurate quantification of libraries by quantitative real-time PCR (qPCR) is important to avoid underperformance or overloading of the sequencers. (6) Assess potential contamination of the RNA as part of the standard post sequencing RNA-Seq QC protocols. Bacterial contamination and genomic DNA contamination are common for FFPE samples due to storage conditions and sample preparation procedures. Samples contaminated with foreign species can waste sequencing coverage, depending on the extent of contamination. In addition, internal contamination can arise from incomplete rRNA depletion, leading to a high percentage of reads mapping to rRNAs. Inefficient genomic DNA removal during DNase digestion could lead to false positive expression detection of transcripts or erroneous de novo assembly of transcripts. Adapter contamination introduced during library preparation is also a common problem for highly degraded RNAs with very short RNA fragments. Contamination can affect the gene and transcript profiling accuracy and lead to false discovery. Therefore, it is important to accurately identify the contamination sources and remove the contamination, if possible, during the sample or library preparation steps, or filter the contaminating reads during the data processing step. (7) Preprocessing and post-alignment quality control are important to detect bad quality and low mRNA content samples. Those samples should be eliminated from further analysis. Gene expression data from samples that generate low gene counts, poor coverage should be used with caution. (8) It is good practice to include biological replicates in order to measure samples variance and correlation to ensure data reproducibility.
FFPE samples represent a very valuable resource for a large number of diseases. The ability to obtain reliable sequence information from such samples would aid a lot of studies aimed at understanding the molecular mechanisms behind various disorders, resistance, and susceptibility. Though the limitations imposed by the frequently suboptimal quality of RNA extracted from such samples do hamper such efforts, the steps described here help to mitigate those limitations to some extent and enable us to make the most of FFPE-RNA to obtain reliable gene expression information.

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>&lt;u&gt;https://www.neb.com/protocols/2017/02/07/protocol-for-use-with-ffpe-rna-nebnext-rrna-depletion-kit&lt;/u&gt;</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 more sensitive to accurately measure the proportion of smaller fragment sizes for highly degraded RNA samples.
In the given sample set, fewer than 10% of the samples (7 of 67) were above the DV200 cut off of 30%, as recommended by Illumina34. About 26% of the samples (19 of 67) had a DV100 &#62; 60% (i.e., higher likelihood of generating good sequence data), 40% (27 of 67) were in the 40%&#8211;60% range for DV100 (i.e., acceptable, but with a lower likelihood of generating good sequence data), and about 10% (7 of 67) had a DV100 of &#60;40% (i.e., very low likelihood of resulting in good sequence data). For 14 of 67 samples, the software was unable to determine the DV values. Table 1 shows a summary of QC metrics for the samples in different DV100 categories. For detailed QC analysis and data correlation for all 67 samples, please see Zhao et al.11.
Given the high degree of degradation in the sample set, a &#8216;total RNA&#8217; library preparation method was chosen, and sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (Table of Materials). In order to improve the representation of the sequencing libraries in spite of the high degree of sample degradation, the maximum possible amount of RNA (1,000 ng when available) was used as input for library preparation. Additionally, the high degradation of the FFPE-RNA samples necessitated the rRNA depletion method, because the degraded transcripts were likely to not have the poly-A tails for mRNA capture. Following the depletion of ribosomal RNA by hybridization to specific probes and digestion of the hybridized transcripts using RNaseH, the remaining transcripts were converted into cDNA using random primers. Size selection was also avoided for libraries prepared from lower input samples. Example traces of final libraries are shown in Figure 3.
Highly degraded FFPE samples represent a great challenge for gene expression profiling in tumor samples. Thus, applying correct bioinformatics analysis methods and software tools is critical to detect artifacts or abnormalities in datasets to ensure high accuracy and reproducibility of gene quantification. The software tools used in this study are listed in the Supplementary Table. In the given sample set, we performed sequencing and library quality assessment, with some example metrics shown in Figure 4. An overview of raw fastq file sequencing quality and sample adapter content are shown in Figure 4A and Figure 4B, respectively. Fastqc screen can help detect contamination, such as bacterial and mouse contamination, in the samples as shown in Figure 4C. In the given sample set, 41 of 67 samples had 5%&#8211;48% bacterial contamination, and six samples had 4%&#8211;11% mouse contamination (Figure 4C). STAR alignment results (Figure 4D) showed the proportion of reads mapped to the reference genome, percentage of reads uniquely mapped to the reference genome, and proportion of reads that were not mapped or mapped to multiple loci. Picard CollectRNAStatistics was used to determine the percent mRNA, intronic, and intergenic bases present in the alignment files (Figure 4E). In order to assess the uniformity of read coverage on gene and transcripts, we used the Picard software tool to generate a gene body coverage plot, which measures the percentage of reads that cover each nucleotide position of all genes scaled into bins from 5&#8242; UTR to 3&#8242; UTR. Figure 4F shows that some degraded libraries had 3&#8217; bias, where more reads are mapped closer to 3&#8217; end than to the 5&#8217; end.
FFPE samples usually have large variability in gene expression profiles that may arise due to variable degradation during sample storage, RNA extraction, or sample processing. It is important to use appropriate statistical methods to uncover the underlying patterns and measure the variation and correlation among samples. We applied Principal Component Analysis (PCA) for six pairs of biological replicates from a subset of the 67 FFPE samples. A PCA plot showed that 26% of total variation was captured by the first principal component and 19% from the second and third components combined (Figure 5). Among the six pairs of replicates, two pairs of replicates had higher variations (correlations below 0.22) than the last four samples (correlation values between 0.7&#8211;0.8) when comparing gene expression values between the replicate pairs. Because the replicates were generated by extracting RNA from two different tissue curls cut from the same FFPE blocks, the tissue age was not a factor in the higher variance here, and it was likely caused by the different amount of bacterial contamination (1%&#8211;55%) as well as different mRNA content (2&#8211;3 fold difference) between the replicates. The randomness of mRNA degradation after extraction could also contribute to the higher variance between samples of similar origin.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61060/61060fig1v2.jpg" /> 
Figure 1: RNaseq analysis workflow.&#160;The flowchart describes the analysis steps for preprocessing, quality assessment, mapping to reference, gene quantification, and differential analysis between different sample groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61060/61060fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61060/61060fig2v2.jpg" /> 
Figure 2: Example Bioanalyzer traces of six different FFPE-RNA samples.&#160;The horizontal axis denotes the molecular weight (bp) and fluorescence units (FU) and the vertical axis shows the concentration of different sized fragments. The RNA Integrity Numbers (RIN), DV200 (i.e., percent of fragments &#62;200 bp), and DV100 (i.e., percent of fragments &#62;100 bp) values are indicated on each profile. A 25 bp peak in each profile indicates the molecular weight marker. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61060/61060fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61060/61060fig3v2.jpg" /> 
Figure 3: Example Bioanalyzer traces of final libraries prepared from four different samples.&#160;The horizontal axis denotes the molecular weight (bp) and fluorescence units (FU) on the vertical axis indicate the concentration of different sized fragments. The lower (35 bp or 50 bp) and upper (10,380 bp) marker peaks are labeled in green and purple, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61060/61060fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61060/61060fig4v2.jpg" /> 
Figure 4: Example multi-QC report for preprocessing QC results.&#160;(A) Line chart showing the percentages of Q30 bases of all sequencing reads in each sample. (B) Sequencing adapter content in raw fastq files. (C) Contamination screen to check closely matched species. (D) Genome mapping statistics. (E) Read distribution based on Gencode gene annotation. (F) Gene body/transcript coverage <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61060/61060fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61060/61060fig5v2.jpg" /> 
Figure 5: Example PCA analysis to show sample group concordance.&#160;PCA analysis for biological replicates. PCA plot with samples plotted in two dimensions using their projections onto the first two principal components. Biological replicates are shown in the same color. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61060/61060fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:font-size="7px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Number of samples</td>
			<td>Median Input for lib prep (ng)</td>
			<td>Median RIN</td>
			<td>Median DV200</td>
			<td>Median DV100</td>
			<td>Median Lib size (bp)</td>
			<td>Median Lib yield (ng)</td>
			<td>Median Lib Molarity (nM)</td>
			<td>Median Specimen storage time (Years)</td>
			<td>Median % contamination</td>
			<td>Median Gene Count</td>
		</tr>
		<tr>
			<td>DV100 &#60;40%</td>
			<td>7</td>
			<td>237.6</td>
			<td>2.5</td>
			<td>6</td>
			<td>34</td>
			<td>445</td>
			<td>24.5</td>
			<td>7</td>
			<td>22</td>
			<td>27.4</td>
			<td>14,759</td>
		</tr>
		<tr>
			<td>DV100 40-60%</td>
			<td>27</td>
			<td>1000</td>
			<td>2.5</td>
			<td>12</td>
			<td>51</td>
			<td>408</td>
			<td>19.8</td>
			<td>5.9</td>
			<td>18</td>
			<td>9.9</td>
			<td>10,202</td>
		</tr>
		<tr>
			<td>DV100 &#62;60%</td>
			<td>19</td>
			<td>1000</td>
			<td>2.3</td>
			<td>26</td>
			<td>73</td>
			<td>355</td>
			<td>84.9</td>
			<td>24</td>
			<td>13</td>
			<td>3.2</td>
			<td>9,993</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of sample set QC metrics. The table shows the QC metrics of the samples, grouped according to their DV100 values. The number of samples in each group is listed, and median values for each metric are shown.
Supplementary Table: Analysis software tools, parameters, and software reference.The table lists the analysis software tools and parameters used in each step of the RNA-seq analysis. The software tool references are listed in the table. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61060/Supplementary_Data.xlsx" target="_blank">Please click here to download this table.</a>

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 ...

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Nanyang Technological University

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12.0K Views.  Frederick National Laboratory for Cancer Research. Ons protocol kan worden gebruikt om de kwaliteit en kwantiteit van genexpressiegegevens gegenereerd uit FFPE-afgeleide RNA-monsters te verbeteren, vooral in suboptimale kwaliteitsmonsters. Deze techniek beoordeelt de RNA-kwaliteit binnen FFPE-weefselmonsters en maakt optimalisatie van hun downstream verwerking mogelijk om de kwantiteit en kwaliteit van de monstersequencinggegevens te verbeteren. Deze methode maakt het mogelijk om een transcriptie in biologisch en klinisch monster volledig te ...

Video: Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

Cancer Research

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Biochemistry

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Biology

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

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