mrna NGS Data Analysis Report

mrna NGS Data Analysis Report Project: Test Project (Ref code: 00001) Customer: Test customer Company/Institute: Exiqon Date: Monday, June 29, 2015 Performed by: XploreRNA Exiqon A/S Company Reg. No. (CVR) 18 98 44 31 Skelstedet 16 DK-2950 Vedbæk Denmark Contact information: exiqon.com/contact

The files listed below can be found in My Projects at XploreRNA Content file_descriptions.html Description Overview of all result files. Data QC All information related to Quality Control of Your samples. This includes QC of individual reads as well as QC of the overall mapping results of all samples. Mapping Results Alignment files (BAM) and alignment index files (BAI) as well as information about splice junctions, deletions, and insertions for each sample. Assembled Transcripts Genes and isoforms identified for each sample including their raw FPKM abundacne estimates. In addition a.cxb file is provided to ease further analysis. Analysis Result file from the analysis of Your samples. This includes: Tables with experession values Differential expression analysis results Unsupervised analysis results Gene Ontology Enrichment analysis Table 1: List of results files. The files can be downloaded from My Projects at XploreRNA. Go to Explore Results and click the download all files link. In the root folder you will find a full description of all files provided, and recommended programs to view the different files. Page 2 of 33

Table of Contents Summary Experimental overview Sample overview Reference genome Work flow Data QC Average Read Quality Average Base Quality Mapping and Yields Results Identified genes Principal Component Analysis Control and Knockdown Heat map and unsupervised clustering Control and Knockdown Identification of novel transcripts Differentially expressed genes, supervised analysis Comparison of knockdown and scramble - Genes Comparison of knockdown and scramble - Isoforms Comparison of knockdown and scramble - Novel isoforms Volcano Plot - Introduction Volcano Plot - knockdown and scramble Gene Ontology Enrichment Analysis - Introduction Gene Ontology Enrichment Analysis knockdown vs scramble Conclusion and next steps Data Analysis workflow Software tools used for the analysis mirsearch References Frequently asked questions Definitions Contact us 4 5 5 5 6 7 7 8 9 11 11 13 14 15 15 16 17 18 18 19 20 21 24 26 26 27 28 29 30 33 Page 3 of 33

Summary Dear Dr. Test customer We have now finalized the Next Generation Sequencing analysis of the transcripts identified in the fastq files you have uploaded to XploreRNA. The principal findings are summarized in this document, including the results of the unsupervised analysis, supervised differential expression analysis and GO enrichment analysis. Additional information, graphs and plots can be found in the files available in My Projects at XploreRNA. The easiest way to identify relevant targets for downstream validation and further study, is to use the Gene Sorting Wizard in My Projects at XploreRNA. With the wizard you can explore each group comparison and sort the full list of differentially expressed transcripts using all relevant criteria, including expression level, fold change, and statistical significance. Once you have selected your candidate genes using the Gene Sorting Wizard, you can with just one click order LNA -enhanced primer sets for qpcr validation, or LNA GapmeRs for efficient antisense inhibition of your mrna or ncrna targets. The designs are done automatically for the sequences identified in Your sample, so there is no need to upload any additional sequences. LNA probes are also available for detection of any mrna or ncrna target by In Situ Hybridization or Northern Blotting. For more information about Exiqon's products for validation and functional analysis of your mrnas or ncrnas of interest, please go to: exiqon.com/rna If you have any questions, please review the Help page XploreRNA.exiqon.com/FAQ Kind regards, Exiqon A/S

Experimental overview Sample overview The table below lists all the samples in this project and their specifications according to the information provided. Sample name Sample groups Sample1 Sample2 Sample3 Sample4 Sample5 knockdown knockdown knockdown control control Table 2: Sample name and sample grouping. Reference genome Annotation of the obtained sequences was performed using the reference annotation listed below. Organism: Homo sapiens Reference genome: GRCh37 Annotation reference: Ensembl Page 5 of 33

Work flow The figure below outlines Exiqon's data analysis pipeline for mrna Next Generation Sequencing. Figure 1: Overview of the mrna NGS data analysis pipeline. Page 6 of 33

Data QC The following sections provide a summary of the QC results obtained for your dataset. This section includes only QC of the reads themselves whereas the subsequent section includes QC of the mapping results. Average Read Quality An overview of the average read quality is shown in Figure 2. Figure 2: Average read quality of the NGS sequencing data. The Q-score is plotted on the x-axis and the density is plotted on the y-axis. A Q-score above 30 is considered high quality data (red dotted line). Read pairs R1 (read1) and R2 (read2) are presented separately for each sample. Page 7 of 33

Average Base Quality An overview of the average base quality is shown in Figure 3. Figure 3: Average base quality of the NGS sequencing data. The Q-score is plotted on the x-axis and the number of bases is plotted on the y-axis. A Q-score above 30 (>99.9% correct) is considered high quality data. Read pairs R1 (read1) and R2 (read2) are presented separately for each sample. Page 8 of 33

Mapping and Yields Mapping of the sequencing data is a useful quality control step in the NGS data analysis pipeline as it can help to evaluate the quality of the samples. Reads are classified into the following classes: Mappable reads: aligning to reference genome Outmapped reads or high abundance reads: For example; rrna, mtrna, polya and PolyC homopolymers Unmapped reads: no alignment possible In a typical experiment it is possible to align 60-90% of the reads to the reference genome, However, this number depends upon multiple factors, including the quality of the sample and the coverage of the relevant reference genome; if the sample RNA was degraded, fewer reads will be mrna specific and more material will be degraded rrna. Table 3 and Figure 4 below summarizes the mapping results. In addition to the mapping results, the table below also shows the total number of reads obtained for each sample. On average 19.7 million reads were obtained for each sample and the average genome mapping rate was 84%. Sample Reads mtrnas rrnas Mapped Unmapped Sample1 23,544,153 0.9% 2.2% 80.9% 16.0% Sample2 20,141,813 0.2% 2.1% 80.3% 17.4% Sample3 17,916,102 0.3% 2.2% 83.1% 14.4% Sample4 21,830,449 0.4% 1.9% 82.1% 15.6% Sample5 15,117,833 0.3% 1.9% 83.4% 14.6% Table 3: Summary of the mapping results for each sample. Page 9 of 33

The following figure summarizes the mapping results for each sample. Figure 4: Summary of mapping results of the reads by sample. Each sample consists of reads that can be classified into the following categories: mapped (reads which align to reference genome), outmapped or high abundance (e.g. rrna, polya, polyc, mtrna) and reads which did not align to anything (unmapped). If you wish to inspect the mapping in detail, please download the BAM alignment files from My Projects at XploreRNA. The detected deletions, insertions, and splice junctions are also available in the Mapping Results folder. Go to Explore Results and click the download all files link. In the root folder you will find a full description of all files provide as well as recommended programs to view the different files. The BAM files can be viewed and inspected in any standard genome viewer such as the IGV browser (Robinson et al.,2011) and (Thorvaldsdóttir et al., 2012) downloadable from https://www.broadinstitute.org/igv/home. Page 10 of 33

Results Below you will find a summary of the principal findings for this project. The complete analysis may be found in the associated files listed in Table 1 on page 2. For a detailed description of the data analysis process see the Work flow section in this report (page 6). Identified genes The number of identified genes per sample was calculated based on alignment to the reference genome. When performing statistical comparisons between groups, we include all genes irrespective of abundance. Ideally, all samples in the study should have similar call rates (similar numbers of genes identified), in order to be comparable. Sample name Sample4 Sample5 Sample1 Sample2 Sample3 Number of Genes identified Number of Isoforms identified 13,712 44,516 13,804 44,173 13,794 44,464 13,866 44,487 13,802 44,187 Table 4: Number of genes and isoforms identified in each sample which have a fragment count estimation of at least 10 counts per gene or isoform. Page 11 of 33

The number of genes identified for each sample based on different fragment count cut-off values is illustrated in the radar plot in Figure 5. The sample name is indicated on the outer rim of the plot. The number of genes identified which have a fragment count estimation of at least 1, 10, 100 or 1000 counts per gene are illustrated as colored rings. If one sample results in a significantly lower number of genes identified at each fragment count cut-off, this is an indication that the sample is deviating from the remaining samples. Ideally, comparable samples should show similar number of genes identified at each fragment count cut-off, resulting in concentric rings of different colors. Figure 5: Radar plot showing number of genes identified for each sample at different fragment count cut-off values. See color scale at top of figure for specification of fragment count cut-off values. Expression levels are measured as FPKM FPKM is a unit of measuring gene expression used for NGS experiments. The number of reads corresponding to the particular gene is normalized to the length of the gene and the total number of mapped reads (Fragments Per Kilobase of transcript per Million mapped reads). In the analysis part the FPKM values are normalized with median of the geometric mean (Anders & Huber, 2010). Page 12 of 33

Principal Component Analysis Control and Knockdown Principal Component Analysis (PCA) is a method used in unsupervised analysis to reduce the dimension of large data sets and is a useful tool to explore sample classes arising naturally based on the expression profile. The 500 genes that have the largest coefficient of variation based on FPKM abundance estimations have been included in the analysis. The figure below represents an overview of how the samples cluster. The largest component in the variation is plotted along the X-axis and the second largest is plotted on the Y-axis. If the biological differences between the samples are pronounced, this will describe the primary components of the variation in the data. This leads to separation of samples in different regions of a PCA plot corresponding to their biology. However, if other factors, e.g. sample quality, introduce more variation in the data, the samples may not cluster according to the biology. Figure 6: Principal component analysis (PCA) plot for Control and Knockdown. The PCA was performed on all samples passing QC using the 500 genes that have the largest coefficient of variation based on FPKM counts. Each circle represents a sample. Based on Normalized FPKM (abundance) for each gene for each sample (genes.fpkm_table can be downloaded from My Projects at XploreRNA). Page 13 of 33

Heat map and unsupervised clustering Control and Knockdown The heat map diagram below shows the result of the two-way hierarchical clustering of genes and samples. It includes the 500 genes that have the largest coefficient of variation based on FPKM counts. Each row represents one gene and each column represents one sample. The color represents the relative expression level of a transcript across all samples. The color scale is shown below: red represents an expression level above the mean; green represents an expression level below the mean. Figure 7: Heat map and unsupervised hierarchical clustering by sample and genes were performed on the listed samples using the 500 genes that have the largest coefficient of variation based on FPKM counts. Data is based on samples from the Control and Knockdown groups. Based on Normalized FPKM (abundance) for each gene for each sample (raw data in genes.fpkm_table can be downloaded from My Projects at XploreRNA). Page 14 of 33

Identification of novel transcripts During the transcriptome assembly process, both known and novel transcripts are identified. A novel transcript is characterized as a transcript which contains features not present in the reference annotation. Thus, a novel transcript can be both a new isoform of a known gene or a transcript without any known features. For example, a novel transcript could be the result of a previously unknown splicing event for a known gene or a previously unknown long noncoding RNA. Identification of novel transcripts depends upon the reference annotation. Transcripts not part of the reference genome used for annotation will be classified as novel. In the results files we classify novel transcripts with known features by listing the known transcripts most closely resembling the novel transcript. For novel transcripts without any known features we provide a locally unique name as transcript identifier. In addition, we provide the genomic positions for the features of the novel transcript, e.g. the location and number of exons. A list of differntially expressed novel isoforms can be found in this report, and the full list of differentially expressed isoforms are available in My Projects at XploreRNA. Go to Explore Results and click the download all files link. In the root folder you will find a full description of all files provide, and recommended programs to view the different files. The table annotations are complex but a good reference is presented in the Cufflinks manual accessible at http://cole-trapnell-lab.github.io/cufflinks/manual/. Differentially expressed genes, supervised analysis To identify differentially expressed genes, it is assumed that the number of reads produced by each transcript is proportional to both its size and abundance. Exiqon Services has customized the analysis pipeline based on the Tuxedo suite, including the Cufflinks, Cuffmerge and Cuffdiff steps of the Tuxedo pipeline. For more details see Data Analysis work flow on page 6. Page 15 of 33

Comparison of knockdown and scramble - Genes The table below shows the individual results for the top 20 most significantly differentially expressed genes. A full list of the differentially expressed genes is available as a.tsv file in My Projects at XploreRNA. Gene ID XLOC_037052 XLOC_027736 XLOC_019256 XLOC_004524 XLOC_030606 XLOC_041844 XLOC_048196 XLOC_007696 XLOC_013906 XLOC_045330 XLOC_012487 XLOC_013938 XLOC_013755 XLOC_029563 XLOC_029829 XLOC_008453 XLOC_024563 XLOC_007499 XLOC_017466 XLOC_008640 Gene Locus knockdown FPKM scramble FPKM Log2 Fold change q_value AREG U6 AC137934.1 PTGS2 BMP2 RP1-67M12.2 ANGPT2 C11orf96 DACH1 KCND2 SLC6A15 EDNRB VWA8 IL1B NR4A2 GRIA4 GDF15 INSC BMF VWA5A 4:75310850-75320726 2:128601137-128615731 16:90252404-90289086 1:186640922-186649559 20:6748310-6760910 6:22113562-22196366 8:6261071-6565730 11:43942608-44022707 13:72012097-72441330 7:119913721-120392568 12:85253491-85307394 13:78469615-78493903 13:42140960-42535256 2:113587327-113594480 2:157180943-157198860 11:105480720-105852819 19:18485540-18499987 11:15133969-15268754 15:40380090-40401093 11:123986068-124018428 63.12 2.63 10.11 43.78 40.34 0.84 1.25 15.23 6.49 2.35 82.28 3.31 112.92 22.76 23.86 12.53 512.04 17.92 24.84 36.14 0.43 171.4 0.18 0.77 0.87 0.02 0.03 0.58 0.25 0.1 3.59 0.15 5.37 1.09 1.23 0.65 26.61 0.95 1.36 2.02-7.21 6.03-5.84-5.83-5.54-5.41-5.21-4.72-4.67-4.53-4.52-4.46-4.39-4.39-4.27-4.27-4.27-4.23-4.19-4.16 0.0122259 Table 5: Genes: Table of the 20 most significantly differentially expressed genes. knockdown and scramble columns are group average FPKM values. FPKM is a unit of measuring gene expression (Fragments Per Kilobase of transcript per Million mapped reads). Transcripts with the highest fold change between groups are shown at the top of the table. Fold change is the log2 fold change of the FPKM between groups knockdown and scramble. q-values shown are p-values that have been adjusted using the Benjamini-Hochberg False Discovery Rate (FDR) approach to correct for multiple testing. As a general guide, fold changes with q-values below 0.05 may be considered significant. The full list of differentially expressed isoforms is available as a.tsv file in My Projects at XploreRNA. Page 16 of 33

Comparison of knockdown and scramble - Isoforms The table below shows the individual results for the top 20 most significantly differentially expressed isoforms (both known and novel). A full list of differentially expressed isoforms is available as a.tsv file in My Projects at XploreRNA. Isoform ID XLOC_037052 XLOC_029829 XLOC_004524 XLOC_008453 XLOC_012487 XLOC_031657 XLOC_012487 XLOC_030606 XLOC_007696 XLOC_019146 XLOC_017466 XLOC_013906 XLOC_013938 XLOC_045330 XLOC_008640 XLOC_029563 XLOC_012487 XLOC_019301 XLOC_014613 XLOC_041488 Gene Locus AREG NR4A2 PTGS2 GRIA4 SLC6A15 FAM65C SLC6A15 BMP2 C11orf96 HSD17B2 BMF DACH1 EDNRB KCND2 VWA5A IL1B SLC6A15 UNKL CDKN3 EBF1 4:75310850-75320726 2:157180943-157198860 1:186640922-186649559 11:105480720-105852819 12:85253491-85307394 20:49202644-49308083 12:85253491-85307394 20:6748310-6760910 11:43942608-44022707 16:82068608-82173236 15:40380090-40401093 13:72012097-72441330 13:78469615-78493903 7:119913721-120392568 11:123986068-124018428 2:113587327-113594480 12:85253491-85307394 16:1401923-1464752 14:54863566-54886936 5:158122927-158526948 knockdown scramble FPKM FPKM 62.98 0.43 17.21 0.17 34.44 0.38 3.13 0.04 12.06 0.17 9.22 0.15 43.31 0.85 40.34 0.87 15.1 0.37 10.88 0.28 12.13 0.32 4.22 0.11 2.57 0.08 1.83 0.06 25.29 0.81 22.28 0.91 22.26 0.95 2.46 0.11 0.45 9.25 1.46 0.07 Log2 Fold Change -7.2-6.69-6.52-6.31-6.15-5.9-5.67-5.54-5.35-5.28-5.24-5.21-5.04-4.99-4.96-4.61-4.56-4.51 4.38-4.33 q_value 0.0298192 0.00443814 0.00443814 0.00955544 0.0073103 0.0290644 Table 6: Isoforms: Table of the 20 most significantly differentially expressed isoforms (known and novel). knockdown and scramble columns are group average FPKM values. FPKM is a unit of measuring gene expression (Fragments Per Kilobase of transcript per Million mapped reads). Isoforms with the highest fold change between groups are shown at the top of the table. Fold change is the log2 fold change of the FPKM between groups knockdown and scramble. q-values shown are p-values that have been adjusted using the Benjamini-Hochberg False Discovery Rate (FDR) approach to correct for multiple testing. As a general guide, fold changes with q-values below 0.05 may be considered significant. The full list of differentially expressed isoforms is available as a.tsv file in My Projects at XploreRNA. Page 17 of 33

Comparison of knockdown and scramble - Novel isoforms The table below lists the top 20 most significantly differentially expressed novel isoforms identified in this project. In the second column in the table below are listed known transcripts most closely resembling the novel transcript. A full list of differentially expressed novel transcripts is available as a.tsv file in My Projects at XploreRNA. Isoform ID XLOC_012487 XLOC_008951 XLOC_034803 XLOC_016569 XLOC_046849 XLOC_013712 XLOC_019977 XLOC_044441 XLOC_031630 XLOC_020238 XLOC_057423 XLOC_010300 XLOC_026275 XLOC_007544 XLOC_039626 XLOC_027344 XLOC_010074 Closest Known Transcript SLC6A15 NLRP10 CLDN11 DLL4 DGKI POSTN CSNK2A2 RBAK SULF2 ZDHHC7 CENPI TEAD4 PSG4 NAV2 RGMB RMND5A PCSK7 XLOC_049974 XLOC_003811 XLOC_000473 LINC00475 PHTF1 EPB41 Locus 12:85253491-85307394 11:7979096-7987016 3:170136652-170578169 15:41221537-41231272 7:137065629-137531838 13:38136718-38172981 16:58163432-58251709 7:5013618-5112854 20:46130552-46415360 16:85007786-85045141 X:100353156-100421146 12:3068495-3154875 19:43696853-43711451 11:19372270-20143148 5:98104353-98134347 2:86730553-87005164 11:117070036117103241 9:94903579-94922203 1:114239452-114302163 1:29213602-29450447 knockdown FPKM 12.06 0.09 1.18 14.41 4.96 104.1 0.57 1.42 4.36 0.51 0.24 0.12 0.62 0.12 4.02 6.11 4.83 scramble FPKM 0.17 1.2 15.47 1.19 0.43 9.75 5.94 0.14 0.43 4.66 2.06 1.05 5.26 1.03 32.76 0.75 38.55 Log2 Fold Change -6.15 3.81 3.72-3.59-3.54-3.42 3.39-3.35-3.32 3.19 3.11 3.11 3.09 3.08 3.03-3.02 3.0 q_value 0.00542043 0.00183482 0.0133829 0.00126576 0.0247324 0.00343073 2.37 2.78 0.26 0.32 0.37 1.85-2.91-2.91 2.86 Table 7: Novel isoforms. Table of the 20 most significantly differentially expressed novel isoforms. knockdown and scramble columns are group average FPKM values. FPKM is a unit of measuring gene expression (Fragments Per Kilobase of transcript per Million mapped reads). Novel transcripts with the highest fold change between groups are shown at the top of the table. Fold change is the log2 fold change of the FPKM between groups knockdown and scramble. q-values shown are pvalues that have been adjusted using the Benjamini-Hochberg False Discovery Rate (FDR) approach to correct for multiple testing. As a general guide, fold changes with q-values below 0.05 may be considered significant. The full list of differentially expressed isoforms is available as a.tsv file in My Projects at XploreRNA. Volcano Plot - Introduction The Volcano plot provides a way to perform a quick visual identification of the genes displaying large-magnitude changes which are also statistically significant. The plot is constructed by plotting -log10(p-value) on the y-axis, and the expression fold change between the two experimental groups on the x-axis. There are two regions of interest in the plot: those points that are found towards the top of the plot (high statistical significance) and at the extreme left or right (strongly down and up-regulated respectively). Page 18 of 33

Volcano Plot - knockdown and scramble Genes that pass the filtering of q-value <0.05 are indicated on the plot (red). For the present study, 6,704 genes pass this filtering. Figure 8: Volcano plot showing the relationship between the p-values and the log2 fold change in normalized expression (FPKM) between knockdown and scramble. Data is based on Normalized FPKM (abundance) for each gene for each sample (gene_exp.diff available in My Projects at XploreRNA). Page 19 of 33

Gene Ontology Enrichment Analysis - Introduction Gene Ontology (GO - Gene Ontology Consortium, 2000) is an initiative to describe genes, gene products and their attributes using vocabulary (GO terms) which is unified and controlled across all species. This enables functional interpretation of experimental data using GO terms, for example via enrichment analysis. We use Gene Ontology enrichment analysis to investigate whether specific GO terms are more likely to be associated with the differentially expressed transcripts. Two different statistical tests are used and compared. Firstly, a standard Fisher s test is used to investigate enrichment of terms between the two groups. Secondly, the Elim method takes a more conservative approach by incorporating the topology of the GO network to compensate for local dependencies between GO which can mask significant GO terms. Comparisons of the predictions from these two methods can highlight truly relevant GO terms. The figure below shows a comparison of the results for the GO (Biological process) terms associated with the significantly differentially expressed transcripts that were identified between the two groups. Complete GO enrichment analysis for all of the comparisons, including Cellular component (CC) and Molecular functions (MF) analysis, is available in My Projects at XploreRNA. Page 20 of 33

Gene Ontology Enrichment Analysis knockdown vs scramble Figure 9: Scatter plot for significantly enriched GO terms associated with genes differentially expressed between knockdown and scramble. Plot shows a comparison of the results obtained by the two statistical tests used. Values along diagonal are consistent between both methods. Values in the bottom left of the plot correspond to the terms with most reliable estimates from both methods. Size of dot is proportional to number of genes mapping to that GO term and coloring represents number of significantly differentially expressed transcripts corresponding to that term with dark red representing more terms and yellow representing fewer. Page 21 of 33

A list of the top 20 significant GO (Biological process) terms is given in the table below. The full list can be obtained through My Projects at XploreRNA (see Table 1 on page 2). GO_ID GO:0051301 GO:0007067 GO:0019048 GO:0000398 GO:0006281 GO:0006355 GO:0000086 GO:0006364 GO:0000184 GO:0006412 GO:0006614 GO:0006414 GO:0006886 GO:0000278 GO:0001701 GO:0000209 GO:0006413 GO:0006271 GO:0006415 GO:0006511 Term Annotated cell division mitosis modulation by virus of host morphology o... mrna splicing, via spliceosome DNA repair regulation of transcription, DNA-depende... G2/M transition of mitotic cell cycle rrna processing nuclear-transcribed mrna catabolic proce... translation SRP-dependent cotranslational protein ta... translational elongation intracellular protein transport mitotic cell cycle in utero embryonic development protein polyubiquitination translational initiation DNA strand elongation involved in DNA re... translational termination ubiquitin-dependent protein catabolic pr... 498 356 346 209 430 3052 147 113 108 484 101 102 823 842 362 164 156 29 86 399 Significant 247 184 129 63 177 975 72 37 33 160 21 26 288 404 141 61 44 22 21 136 Expected p-value 145.3 103.87 100.95 60.98 125.46 890.48 42.89 32.97 31.51 141.22 29.47 29.76 240.13 245.67 105.62 47.85 45.52 8.46 25.09 116.42 3.0e-19 1.5e-14 2.9e-14 2.5e-13 2.7e-13 1.2e-12 2.2e-12 3.1e-11 3.4e-10 6.0e-10 6.2e-09 1.6e-08 2.6e-08 3.7e-08 6.3e-08 6.4e-08 1.3e-07 1.3e-07 1.6e-07 2.5e-07 Table 8: The top 20 significant GO (Biological Process) terms associated with transcripts found to be differentially expressed between knockdown and scramble. To illustrate how the different GO terms are linked, a GO network has been created. The network is shown below and networks of varying complexity are available through My Projects at XploreRNA. Page 22 of 33

Figure 10: GO network generated for the enriched GO terms (Biological Process) associated with genes differentially expressed between knockdown and scramble. Nodes are colored from red to yellow with the node with the strongest support colored red and nodes with no significant enrichment colored yellow. The five nodes with strongest support are marked with rectangular nodes. A high-resolution version of this figure is available in My Projects at XploreRNA Page 23 of 33

Conclusion and next steps The analysis of your RNA Next Generation Sequencing data has been completed. There are many considerations when deciding which of the differentially expressed transcripts to validate and study further. First of all it is important to be sure that the NGS dataset is of good quality, and that there are no sources of experimental or technical variation that may confound the results. Data quality Ensure that the data for all samples passed all of the QC metrics, with a high Q-score, indicating good technical performance of the NGS experiment. A high percentage of the reads should be mappable to the reference genome, indicating high quality samples. Do samples cluster according to biological groups? The unsupervised analysis will reveal if the samples cluster according to their biological groups, indicating that the sample groups are responsible for the largest variation on the samples. If the samples do not cluster according to biological groups, consider any other possible sources of experimental or technical variation in the experiment. Use the Gene Sorting Wizard at XploreRNA to select candidates for downstream validation The easiest way to select your candidates for validation and further study is using the Gen Sorting Wizard in My Projects at XploreRNA. There you can explore each group comparison and sort the full list of differentially expressed genes using all relevant criteria, including expression level, fold change, and statistical significance. Below are some general considerations when selecting candidates for validation and further study. Fold change. Smaller fold changes tend to be more affected by technical variance, and hence may be at greater risk of false-positive signals. To study transcripts with small fold changes, considerably more technical or biological replicates should be included in the validation. Expression level. When navigating through these data, counts lower than 1-5 FPKM (on average) per group might be difficult to validate in a qpcr experiment. Statistical significance. Fold changes with adjusted p-values (q-values) below 0.05 may be considered significant, and have a greater chance of being validated by qpcr. Novel transcripts. Many of the novel transcripts identified may be novel isoforms of known transcripts, or alternative start sites of known transcripts. Reference genes. NGS data can also be used to identify stably expressed transcripts that may be used as reference genes for normalization in qpcr validation experiments. Genes that are stably expressed across all samples can be identified using e.g. NormFinder (www.mdl.dk) or genorm (Vandesompele et al., 2002).

Validating your NGS resultsqpcr Ordering qpcr primers for your candidate genes could not be easier. Simply select your shortlist of genes for validation using the Gene Sorting Wizard at XploreRNA, click design qpcr primers and use Exiqon's web tool to design LNA -enhanced primer sets for your mrnas or ncrnas of interest. The design is done automatically, so there is no need to upload any sequences. For more information about Exiqon's LNA qpcr system, please go to Exiqon custom LNA qpcr: ISH and Northern blotting. LNA detection probes are available for any mrna or ncrna. For more information please go to Exiqon mrna ISH Functional Analysis Silencing your candidate genes. You can easily order Antisense LNA GapmeRs for potent and specific knockdown of your candidate genes. Simply select your shortlist of genes using the Gene Sorting Wizard at XploreRNA, click design GapmeRs and use Exiqon's online tool to design LNA GapmeRs for your mrnas or ncrnas of interest. The design is done automatically, so there is no need to upload any sequences. For more information about Exiqon's LNA GapmeRs, please go to Exiqon GapmeRs microrna regulation. Find out which micrornas are known or predicted to regulate your candidate genes. Simply search using the gene name at mirsearch. For more information about mirsearch, please see page 27.

Data Analysis workflow Software tools used for the analysis Our NGS data analysis pipeline is based on the Tuxedo software package, which is a combination of open-source software, and implements peer-reviewed statistical methods. In addition we employ specialized software developed internally at Exiqon to interpret and improve the readability of the final results. The components of our NGS data analysis pipeline for RNA-seq include Bowtie2 (v. 2.2.2, see Langmead B and Salzberg S. (2012) ), Tophat (v2.0.11, see Trapnell, C., et al. (2009) ) and Cufflinks (v2.2.1, see Trapnell, C., et al. (2010) and Trapnell, C., et al. (2012)), and are described in detail below. Tophat is a fast splice junction mapper for RNA-seq reads. It aligns the sequencing reads to the reference genome using the sequence aligner Bowtie2. Tophat also uses the sequence alignments to identify splice junctions for both known and novel transcripts as well as identification of insertions and deletions. Cufflinks takes the alignment results from Tophat and assembles the aligned sequences into transcripts, thereby constructing a map or a snapshot of the transcriptome. To guide the assembly process, an existing transcript annotation is used (RABT assembly). In addition, we perform fragment bias correction which seeks to correct for sequence bias during library preparation (see Kasper et al., 2010 and Adam et al., 2011). The Cufflinks assembles aligned reads into different transcript isoforms based on exon usage and also determines the transcriptional start sites (TSSs). When comparing groups, Cuffdiff is used to calculate the FPKM (number of fragments per kilobase of transcript per million mapped fragments) and test for differential expression and regulation among the assembled transcripts across the submitted samples using the Cufflinks output. Cuffdiff can be used to test differential expression at different levels, from CDS and gene specific, down to the isoform and TSS transcript level. For more information on the Cuffdiff module, see Trapnell et al., (2013). As a final step custom software is used for post processing of Cuffnorm and Cuffdiff results. We use these tools to generate a visual representation of your sequencing results to aid the interpretation of the sequencing data and the analysis results. Page 26 of 33

mirsearch If you are interested in finding out which micrornas are regulating your transcripts, Exiqon offers two options: mirsearch 3.0 An interactive mirsearch database, offering you up-to-date information on micrornas, their target genes as well as expression and disease associations, supported by references with integrated access to PubMed. mirsearch includes a built-in report feature which allows you to easily collect and store all the relevant information gathered. Search using a gene name or keyword and find: micrornas that regulate specific genes (validated and predicted interactions) Search using a microrna name and find: Regulated genes (validated and predicted interactions) Potentially co-transcribed micrornas Diseases in which the microrna has been shown to be regulated Tissues/samples in which the microrna has been found XploreRNA XploreRNA is an advanced database search tool for scientists engaged in transcriptome analysis. The XploreRNA App enables access to relevant public and proprietary genetic and molecular biology databases through a simple user interface. All databases are cross-annotated and regularly updated by advanced text mining of the literature. The app provides information from major databases such as Ensembl and mirbase. All search results provide information on literature references with integrated access to PubMed. The XploreRNA App can be downloaded from App Store and Google Play. Page 27 of 33

References Anders S. and Huber W. (2010) Differential expression analysis for sequence count data. Genome Biology 11: R106 Benjamini and Hochberg (1995).Journal of the Royal Statistical Society Series B, *57*, 289-300. Kasper D., et al. (2010), Biases in Illumina transcriptome sequencing caused by random hexamer priming Nucleic Acids Research, Volume 38, Issue 12. Kellis, M., et al.(2013) Defining functional DNA elements in the human genome. PNAS, Vol. 111:6131-6138. Langmead B, Salzberg S. (2012), Fast gapped-read alignment with Bowtie 2. Nature Methods. 9:357-359. Marinov, G. K., et al (2014) From single-cell to cell-pool transcriptomes: Stochasticity in gene expression and RNA splicing. Genome Res. 24: 496-510. Roberts, A., et al. (2011) Identification of novel transcripts in annotated genomes using RNASeq. Bioinformatics, 27(17): 2325-2329. Roberts, A., et al., (2011) Improving RNA-Seq expression estimates by correcting for fragment bias Genome Biology, Volume 12, R22. Robinson, J.T., et al (2011) Integrative Genomics Viewer. Nature Biotechnology 29,24 26. Thorvaldsdóttir, H., et al. (2012) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Briefings in Bioinformatics. Trapnell, C., et al. (2010). Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology, 28(5): 511-515. Trapnell,C., et al.(2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols 7,562 578 Trapnell, C., et al. (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics (Oxford, England), 25(9):1105-1111 Vandesompele et al., (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3(7):research0034.1 0034.11 Page 28 of 33

Frequently asked questions What is Q-score? Answer: A quality score (or Q-score) is an estimation of the probability of an incorrect base call. Q-score = -10 log10(p) where p is the estimated probability of the base call being wrong. A quality score of 10 indicates an error probability of 0.1, a quality score of 20 indicates an error probability of 0.01, a quality score of 30 indicates an error probability of 0.001, and so on. A Q-score above 30 (>99.9% correct) is considered high data quality. In order to pass the Data QC, all samples must have an average Q-score across both the first halv and the second half of the reads of at least 16 (>97.5% correct). Question: What is the difference between FPKM and RPKM? Answer: RPKM stands for Reads per Kilobase of transcript per Million mapped reads. FPKM stands for Fragments per Kilobase of transcript per Million mapped fragments. The term fragments refers to the cdna fragments present during library preparation. Both RPKM and FPKM are normalized numbers which tell you something about the relative abundance of, for example, an assembled transcript. In paired-end sequencing, two reads are produced per cdna fragment during library preparation, whereas only one read is produced per cdna fragment in single-end sequencing. Thus, single-end versus paired-end sequencing will affect the value of RPKM but not FPKM. Consequently, FPKM is preferred over RPKM as it will provide values comparable between single-end sequencing and paired-end sequencing Question: What does 1 FPKM mean in terms of abundance? Answer: This is difficult to estimate and highly variable according to cell type and the total number of mrnas in a given cell. For example, It was estimated that in a single cell analysis of the cell line GM12878, that one transcript copy corresponds to 10 FPKM (Marinov 2014). Others find that FPKMs are not directly comparable among different subcellular fractions, as they reflect relative abundances within a fraction rather than average absolute transcript copy numbers per cell (Kellis 2013). Depending on the total amount of RNA in a cell, one transcript copy per cell corresponds to between 0.5 and 5 FPKM in PolyA+ whole-cell samples according to current estimates with the upper end of that range corresponding to small cells with little RNA and vice versa. Question: What is a novel RNA transcript? Answer: A novel transcript is characterized as a transcript from a region that lacks annotation not present in the reference annotation. Identification of novel transcripts depends therefore in the reference annotation. Question: A novel transcript identified seems to be a known gene when I look it up in the gene browser, why is that? Answer: Most novel transcripts are not new genes but different isoforms of previously annotated genes. A novel transcript is most commonly a novel combination of exons or a different start site. Page 29 of 33

Definitions Gene The standard definition of a gene is a high level feature on the genome that codes for a protein or RNA with a function in the organism The same gene may encode multiple different RNA transcripts (or isoforms) through alternative splicing or different transcriptional start sites. In the context of RNA-seq, the term gene is used to refer to all RNA transcripts (or isoforms) encoded by the same gene. For example, when analyzing differentially expressed genes, the reads from all transcripts derived from the same gene are included. Gene_ID Gene identifier. For known genes this will be the gene id from the annotation source, e.g. an Ensembl gene ID. For novel genes, this will be a unique generic identifier, e.g. CUFF.2 Transcript RNA sequence (e.g. mrna, ncrna, trna or rrna) transcribed from DNA.Transcripts may also be referred to as isoforms. Transcript ID Transcript identifier. For known genes this will be the transcript id from the annotation source, e.g. an Ensembl gene ID. For novel transcripts, this will be a generic identifier, e.g. CUFF.2.1 Primary transcript RNA sequence transcribed from DNA. The primary transcript is then processed (e.g. by addition of 5' cap, 3'-polyadenylation, alternative splicing) to yield various mature RNA products such as mrnas, ncrnas, trnas, and rrnas. Multiple primary transcripts may be transcribed from the same gene by use of different transcriptional start sites. Isoforms Different closely related transcripts arising from the same primary transcript (and same gene or DNA sequence) by alternative splicing of exons for example. Isoforms may also be referred to as transcripts. Novel mrna A transcript which contains features not present in the reference annotation. A novel transcript can be both a new isoform of a known gene or a transcript without any known features. A novel transcript is most commonly a novel combination of exons or a different start site. TSS Transcriptional Start Site Page 30 of 33

TSS_ID A unique identifier for the inferred Transcriptional Start Site (TSS). Note: this identifier is unique only within a single analysis. CDS Coding DNA Sequence Promoter A region of DNA that initiates transcription of a particular gene. Promoters are located near the transcriptional start sites of genes. P_ID Promoter ID. This value is extracted from reference annotations, which contain CDS information. GO Gene Ontology (GO - Gene Ontology Consortium, 2000) is an initiative to describe genes, gene products and their attributes using vocabulary (GO terms) which is unified and controlled across all species. The GO terms are categorized into three GO domains: molecular function, biological process and cellular component. GO_ID Unique identifier for a Gene Ontology (GO) term. Exon Sequence that remains present within the final mature RNA product of that gene after introns have been removed by splicing Intron A sequence within a gene that is removed by splicing during maturation of the final RNA product. Outmapped reads or high abundance reads For example; rrna, mtrna, polya and PolyC homopolymers Unmapped reads No alignment to the reference genome is possible. Explanations for this include the read being too short, low read quality or contaminations. Mappable reads Sequences which can be aligned to the reference genome trna Transfer RNA rrna ribosomal RNA mtrna mitochondrial RNA phix Libraries generated from the PhiX virus used as a control in sequencing runs Q-score Quality score used to assess the quality of the bases or reads in NGS data. See FAQs for further explanation. Page 31 of 33

q-value p-values that have been adjusted to correct for multiple testing FPKM Fragments per Kilobase of transcript per Million mapped fragments Reads DNA Sequence generated by the sequencing machine (for paired end sequencing the same strand is sequenced in both directions forward and reverse) Fragment Count The number of fragments originating from a feature Page 32 of 33

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