Illumina RNA Prep with Enrichment a Fast, Integrated Workflow for RNA Enrichment Applications

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

re e ia Prep i rie 10000 1 RNA quantitation 1 RNA quantitation and quali cation and quali cation 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, quanti cation, and normalization 10 5 Ligation e a r 5 Single, 90 min enrichment Capture, washes, and elution

PCR, quanti cation, and UHR, 100 ng input 6 6 PCR ampli cation of 0 normalization enriched library re a ay re 7 First enrichment 7 Quanti cation 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 ampli cation of UHR, 10 ng input enriched library ereae i 10 Quanti cation and a aay ie 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 from Illumina RNA Prep with Exome (Figure 6). Enrichment libraries to data from TruSeq RNA Exome libraries, a These results demonstrate that Illumina RNA Prep with Enrichment standard solution for RNA enrichment, showed high concordance provides high capture efficiencies that focus sequencing efforts on (Figure 7). Of note, the sigmoid shape of the data plot is the result the high value content of RNA coding regions. By working with more of index hopping from adapters used with TruSeq RNA Exome. focused content, Illumina RNA Prep with Enrichment requires lower Importantly, Illumina RNA Prep with Enrichment uses unique dual sequencing depths and produces smaller data sets that result in time indexes (UDIs), which are designed to eliminate index recombination. and cost savings.

chr12

p13.32 p13.2 p12.3 p12.1 p11.22 p11.1 q12 q13.11 q13.2 q14.1 q14.3 q21.1 q21.31 q21.33 q23.1 q23.3 q24.12 q24.23 q24.32

5,121 bp

6,643,000 bp 6,644,000 bp 6,645,000 bp 6,646,000 bp 6,647,000 bp 6,648,000 bp

[0 - 651] U1_FFPE coverage

FFPE 20ng

GAPDH

RefSeq Genes GAPDH

GAPDH

GAPDH Figure 5: Coverage of coding regions Illumina RNA Prep with Enrichment—A library prepared from 20 ng of low-quality FFPE sample and enriched with the Illumina Exome Panel was sequenced at 25M reads. Coverage of the GAPDH control gene using Broad IGV is displayed, showing reads aligning across coding exons and demonstrating good target capture.

For Research Use Only. Not for use with diagnostic procedures. 470-2020-001-A | 3 Design Content | Prepare Library | Sequence | Analyze Data

the novel strain of coronavirus (CoV) responsible for the COVID-19 pandemic. Validation and protocol adjustments may be required when 10 r2 = 0.923 combining Illumina RNA Prep with Enrichment and a custom panel.

5 Summary Illumina RNA Prep with Enrichment offers a streamlined solution and 0 simple, rapid workflow for targeted RNA-Seq. It offers extraordinary flexibility for input type, including degraded samples, and supports low

log2 fold change -5 input amounts. The modular design supports a wide range of RNA-

TruSeq RNA Exome TruSeq Seq applications across regions of interest, eg with the Illumina Exome Panel and Respiratory Virus Oligos Panel, enabling detection and -10 discovery studies such as allele-specific expression, fusion detection, biomarker screening, and more. -10 -5 0 5 10 15 log2 fold change Illumina RNA Prep with Enrichment Learn more To learn more about Illumina RNA Prep with Enrichment and view Figure 7: Concordance with TruSeq RNA Exome—Illumina RNA Prep with Enrichment Plot shows high concordance with TruSeq RNA Exome, as currently available options for panel content, visit www.illumina. measured by log2 fold change for UHR RNA (Agilent, Catalog no. 740000) vs. com/products/by-type/sequencing-kits/library-prep-kits/rna-prep- Human Brain Total RNA (Thermo-Fisher, Catalog no. AM7962). All libraries were enrichment.html prepared from 10 ng input. TruSeq RNA Exome libraries were enriched as a 4-plex, Illumina RNA Prep with Enrichment libraries were enriched as a 3-plex. All data is downsampled to 25M clusters per library. Data was analyzed with the Ordering information BaseSpace Cufflinks Assembly & DE App v 2.1.0. Library prep Catalog no. Illumina RNA Prep with Enrichment, 20040536 Flexible and scalable throughput (L) Tagmentation (16 samples, 1-plex)a By combining Illumina RNA Prep with Enrichment and high-throughput Illumina RNA Prep with Enrichment, 20040537 instruments such as the NextSeq™ 550 and NovaSeq 6000 Systems, (L) Tagmentation (96 samples, 3-plex)a laboratories can sequence significantly more samples per run without Indexes Catalog no. compromising data quality. For additional increases in sample IDT for Illumina DNA/RNA UD Indexes Set A, 20027213 throughput, Illumina RNA Prep with Enrichment supports multiplexing Tagmentation (96 indexes, 96 samples)b * with 384 unique dual indexes (UDIs). In addition to eliminating index IDT for Illumina DNA/RNA UD Indexes Set B, 20027214 misassignment, UDIs help to decrease sequencing costs by allowing Tagmentation (96 indexes, 96 samples)b up to 384 samples to be loaded on a single NovaSeq S4 flow cell for IDT for Illumina DNA/RNA UD Indexes Set C, 20042666 significantly increased throughput. Tagmentation (96 indexes, 96 samples) Available soon IDT for Illumina DNA/RNA UD Indexes Set D, 20042667 Modular design for a broad range of RNA Tagmentation (96 indexes, 96 samples) Available soon applications Enrichment panel Catalog no. Illumina Exome Panel 20020183 By combining RNA library preparation and enrichment performance 20044312 with the proven accuracy of Illumina sequencing by synthesis (SBS) Illumina Respiratory Virus Oligos Panel v2 Available soon chemistry,2 Illumina RNA Prep with Enrichment supports both fixed and custom panels of varying sizes for advanced study designs in a. The 16 sample kit has enough reagents for 1-plex, 16 enrichment reactions, the 96 sample kit has enough reagents for 3-plex, 32 enrichment reactions. a variety of areas. Examples include the Illumina Exome Panel for b. “IDT for Illumina DNA/RNA UD Indexes” are new names for “IDT for Illumina Nextera analysis of coding regions of the transcriptome and the more focused DNA UD Indexes”; kit contents remain the same. Respiratory Virus Oligos Panel, which features ~7800 probes designed to detect respiratory viruses, recent flu strains, and SARS-CoV-2, References 1. von Ahlfen S, Missel A, Bendrat K, and Schlimpberger M. Determinants of

* Up to 192 UDIs will be supported at product launch. Additional UDIs will be available later RNA quality from FFPE samples. PLoS ONE. 2007;2(12): e1261. in 2020. 2. Bentley DR, Balasubramanian S, Swerdlow HP, et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 2008;456:53-59.

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