Design Content | Prepare Library | Sequence | Analyze Data Illumina RNA Prep with Enrichment A fast, integrated workflow for RNA enrichment applications. Highlights First- and second-strand cDNA synthesis • Fast and simple RNA enrichment workflow Prepare libraries in as little as nine hours with less than two cDNA hours of hands-on time • Exceptional data quality from difficult samples Achieve high sensitivity from as little as 10 ng total RNA from eBLTLeBLT eBLTL Tagmentation fresh or frozen samples or 20 ng total RNA from degraded FFPE samples Enrichment Bead-Linked cDNA cDNA-eBLT-L complex Transposome (eBLTL) • Focused and affordable RNA sequencing Tagment with eBLTL Maximize discovery power with robust capture efficiency at reduced sequencing depth for accurate, unbiased detection Tagmented library • Flexible and scalable throughput Index PCR primers p5 Multiplex up to 384 samples in a single run with unique dual Index2 Read 1 sequencing primer (Rd1 SP) indexes Read 2 sequencing primer (Rd2 SP) Index1 p7 Index PCR Introduction p5 Index2 Rd1 SP Rd2 SP Index1 p7 Indexed library RNA sequencing (RNA-Seq) with next-generation sequencing (NGS) Pool pre-enriched libraries (optional) is a powerful method for discovering, profiling, and quantifying RNA transcripts. Advantages of RNA-Seq include: Pooled sample library • Targeted RNA-Seq analyzes expression in a focused set of Add biotinylated probe panel genes. Enrichment enables cost-effective RNA exome analysis using sequence-specific capture of the coding regions of the transcriptome. It is ideal for low-quality, formalin-fixed paraffin- embedded (FFPE) samples. Enrich using streptavidin magnetic beads • Total RNA-Seq provides an unbiased, hypothesis-free approach for comprehensive analysis of the transcriptome. It accurately measures gene and transcript abundance and detects both known and novel features in coding and multiple forms of noncoding RNA. Enriched library • Messenger RNA (mRNA)-Seq sensitively and accurately quantifies Elute enriched library from beads gene expression, identifies known and novel isoforms in the coding Enriched and indexed library ready for sequencing transcriptome and measures allele-specific expression. Figure 1: Illumina RNA Prep with Enrichment chemistry—After cDNA Illumina RNA Prep with Enrichment, (L) Tagmentation offers a synthesis, a uniform tagmentation reaction mediated by eBLT-Ls followed by a streamlined solution for targeted RNA-Seq. It offers high flexibility single, 90-minute hybridization reaction enables a fast and flexible workflow. regarding input type and amount to provide a wide range of RNA-Seq applications, enabling detection and discovery studies such as allele- enrichment Bead-Linked Transposomes (eBLT) optimized for RNA specific expression, fusion detection, biomarker screening, and more. (eBLTL) that mediate a uniform tagmentation reaction, eliminating Combining Illumina RNA Prep with Enrichment with the Illumina Exome the need for separate fragmentation steps to save time. Combined Panel provides a comprehensive view of the coding transcriptome for with innovations to the hybridization reaction, the workflow features maximum discovery power at a fraction of the sequencing depth. fewer steps, shorter incubation times, and numerous safe stopping points and a total assay time that is > 50% faster than TruSeq™ RNA Fast and simple RNA enrichment workflow Exome (Figure 2). In addition to manual preparation, Illumina RNA Prep with Enrichment is designed to be compatible with liquid-handling Illumina RNA Prep with Enrichment uses On-Bead Tagmentation platforms for an automated workflow, providing highly reproducible followed by a simplified, single 90-minute hybridization step to provide sample handling, reduced risk of human error, and less hands-on time. a rapid workflow (Figure 1). On-Bead Tagmentation features For Research Use Only. Not for use with diagnostic procedures. 470-2020-001-A | 1 Design Content | Prepare Library | Sequence | Analyze Data TruSeq RNA Exome Illumina RNA Prep with Enrichment 10000 1 RNA quantitation 1 RNA quantitation and qualication and qualication Fragmentation is not required 2 2 RNA fragmentation 2 cDNA synthesis 1000 R = 0.99 3 cDNA synthesis 3 Tagmentation using BLT chemistry 100 4 A-tailing 4 PCR, quantication, and normalization 10 5 Ligation less than 9 hours 5 Single, 90 min enrichment Capture, washes, and elution PCR, quantication, and UHR, 100 ng input 6 6 PCR amplication of 0 normalization enriched library more than 2 days more 7 First enrichment 7 Quantication and Capture, washes, and elution normalization 0.1 8 Second enrichment 8 Ready for sequencing Capture, washes, and elution 0.1 0 10 100 1000 10000 9 PCR amplication of UHR, 10 ng input enriched library > 50% decrease in 10 Quantication and total assay time normalization Figure 3: High-quality data from low input samples—Illumina RNA Prep with 11 Ready for sequencing Enrichment achieves high data concordance between input amounts of 10 ng and 100 ng total RNA from universal human reference (UHR). UHR RNA libraries were sequenced on a NovaSeq 6000 System, subsampled to 25M clusters per Figure 2: Illumina RNA Prep with Enrichment delivers a fast workflow— library. Data was analyzed with the BaseSpace RNA-Seq Alignment v 1.1.1 app. On-Bead Tagmentation and a single, 90-minute hybridization step combine to deliver a faster workflow with fewer steps compared to TruSeq RNA Exome. chr22 chr9 High-quality data chr22:23,629,909-23,639,668 chr9:133,724,597-133,734,356 Highly accurate data from low-input and FFPE samples [0 - 913] [0 - 913] U1_K562...overage High capture efficiency and coverage uniformity minimize the required U1_K562_10ng_Lot2_CE sequencing depth to determine expression levels accuratelyX_1plex_Rep125M.alig and without bias. Starting with as little as 10 ng total RNA,nments.bam Illumina RNA Prep with Enrichment produces quality data with high concordance [0 - 134] [0 - 134] U1_K562...overage between varying amounts of input RNA from fresh or frozen samples (Figure 3). Precious tumor–normal specimens or FFPEU1_K562_10ng_Lot2_CE archival tissue samples provide a rich source of biological informationX_1plex_Rep125M.alig for gene nments.SA.bam expression profiling; however, they can be difficult to study due to nucleic acid degradation from the fixation and storageSequence process.1 RefSeq Genes Illumina RNA Prep with Enrichment produces quality data with BCR ABL1 input as low as 20ng of input RNA from FFPE samples. Together, these results demonstrate that Illumina RNA Prep with Enrichment Figure 4: Detection of BCR-ABL1 gene fusion—Libraries prepared from 10 ng is an ideal solution for precious or degraded samples with limited K-562 cell line RNA using Illumina RNA Prep with Enrichment and the Illumina Exome Panel resulted in successful detection of the BCR-ABL1 gene fusion, starting material. using the Broad Integrative Genomics Viewer (IGV). Top alignment track shows all reads; bottom track shows only reads supporting the BCR-ABL1 fusion. Gene fusion detection in low-input and FFPE samples To demonstrate the ability of Illumina RNA Prep with Enrichment to Table 1: Gene fusion detection recognize structural variants within RNA transcripts, fresh/frozen RNA Fusion (source) RIN Detection and FFPE samples were enriched using the Illumina Exome Panel input ™ and sequenced using the NovaSeq 6000 System. Results showed BCR-ABL1 (K-562) 7.4 10 ng 6/6 replicates (100%) a 100% call rate for BCR-ABL1 (Figure 4) and TPM3-NTRK1 gene TPM3-NTRK1 (colorectal cancer) 2.5 20 ng 6/6 replicates (100%) fusions across six replicates of the K-562 cell line (RNA integrity number, RIN = 7.4, DV200 = 90%) and a colorectal cancer cell line (RIN = 2.5, DV200 = 85%), respectively (Table 1). 2 | 470-2020-001-A For Research Use Only. Not for use with diagnostic procedures. Design Content | Prepare Library | Sequence | Analyze Data Focused, affordable RNA-Seq Illumina RNA Prep with Enrichment UHR Exceptional exonic coverage FFPE Illumina RNA Prep with Enrichment can be used with the Illumina Exome Panel, which features a highly optimized probe set that delivers TruSeq RNA Exome comprehensive coverage of coding RNA sequences (Table 2). UHR Table 2: Illumina Exome Panel specifications FFPE Coverage specification Illumina Exome Panel No. of target genes 21,415 0% 25% 50% 75% 100% No. of target exonic regions 214,126 % Bases aligned to region No. of probes 425,437 RefSeq exome percent covered 98.3% Coding UTR Intron Intergenic To evaluate the performance of Illumina RNA Prep with Enrichment Figure 6: Coverage of coding regions with Illumina RNA Prep with for exome sequencing, libraries were prepared from universal human Enrichment—Libraries prepared from 10 ng UHR RNA and 20 ng FFPE RNA reference (UHR) RNA and FFPE RNA using Illumina RNA Prep with using Illumina RNA Prep with Enrichment and the Illumina Exome Panel show Enrichment. Resulting libraries were sequenced on a NovaSeq 6000 more than 85% of the data aligned to coding and UTRs. Data from TruSeq RNA Exome libraries are shown for comparison. Libraries were sequenced on a System at 2 × 100 bp (25 M reads). Data analysis with the Enrichment NovaSeq 6000 System at 2 × 100 bp, subsampled to 25 M reads. App in BaseSpace™ Sequence Hub revealed that Illumina RNA Prep with Enrichment resulted exceptional exonic coverage (Figure 5) with > 85% of the bases covered aligning to coding sequence and Concordance with TruSeq RNA Exome untranslated regions (UTR) of RNA, comparable to TruSeq RNA Comparison of data generated