KAPA STRANDED RNA-SEQ KIT WITH RIBOERASE:

Transcription

1 Poster Note As presented at AGBT 5, Marco Island, FL KAPA STRANDED RNA-SEQ KIT WITH RIBOERASE: An effective and reliable enzymatic method for the depletion of ribosomal RNA for preparation of high-quality libraries for transcriptome sequencing. Authors Martin Ranik, Ross Wadsworth, Piet Jones, Jennifer Pavlica, Deepti Sanjai, Rachel Kasinskas and Eric van der Walt

2 INTRODUCTION Ribosomal RNA (rrna) accounts for the vast majority of total cellular RNA, and its efficient removal is critical during the RNA-Seq library preparation process in order to maximize the yield of biologically-informative transcriptome-derived reads. Sequencing of total RNA samples that have been depleted of rrna provides a more comprehensive representation of the transcriptome compared to mrna sequencing. We have developed a flexible and highly effective method for targeted enzymatic depletion of rrna using complementary oligonucleotide probes and RNase H degradation. Our method delivers a more effective and automationfriendly rrna depletion workflow compared to traditional bead-based capture methods, and is compatible with human, mouse, rat and non-human primate samples. To demonstrate this, we provided independent laboratories with the same Universal Human Reference RNA sample (UHR) to prepare libraries using the KAPA Stranded RNA-Seq Kit with RiboErase (Human/Mouse/Rat) and sequence using an Illumina HiSeq. Analysis of the rrna depleted sequencing data revealed high reproducibility, sensitivity, and coverage of the whole transcriptome. KAPA RiboErase is the rrna depletion module within the KAPA Stranded RNA- Seq Kit with RiboErase

3 Kapa Biosystems has developed a flexible and highly effective method for targeted enzymatic depletion of rrna using complementary oligonucleotide probes and RNase H degradation. In the KAPA RiboErase workflow, cytoplasmic (5S, 5.8S, 8S, and 8S) and mitochondrial (S and 6S) rrna from human, mouse or rat samples are efficiently targeted for removal from. - µg of total RNA by hybridizing complementary oligonucleotides. FIGURE KAPA Stranded RNA-Seq Kit with RiboErase workflow Hybridize RNA Deplete rrna Bead Cleanup DNase Digestion Hybridize complementary oligonucleotides targeting the rrna. Treatment with RNase H degrades rrna targeted by complementary oligonucleotides. Removal of degraded rrna and RNase H prevents off-target depletion. Degrades complementary oligonucleotides to prevent carryover into library preparation. Hybridization of complementary oligonucleotides targets rrna from human, mouse or rat samples for near complete removal from. - µg of total RNA. The workflow is optimized to be automation friendly, with pipetting volumes compatible with most automated platforms. Targeted enzymatic degradation of the rrna prevents accidental carryover of concentrated rrna, a high-risk feature of capture bead-based methods. The rrna depleted sample can be eluted in RNasefree water or X Frag, Prime and Elute Buffer from the KAPA Stranded RNA-Seq Kit for a streamlined and efficient workflow for library preparation. The RNA-Seq protocol utilizes KAPA HiFi DNA Polymerase for high-efficiency and low-bias library amplification, ensuring a more complete representation of difficult (e.g., high GC) transcript regions. Bead Cleanup Removal of DNase and degraded oligonucleotides. Library Preparation Construction of libraries according to the KAPA Stranded RNA-Seq Kit protocol.

4 rrna DEPLETION PROVIDES MOST COMPREHENSIVE VIEW OF TRANSCRIPTOME FIGURE Summary of rrna depletion data % Intergenic Rate Intronic Rate 9% Exonic Rate 8% rrna 7% KAPA Stranded RNA-Seq Kit with RiboErase removes the majority of cytoplasmic and mitochondrial rrna from total RNA samples. The resulting transcriptome sequence contains a greater proportion of reads derived from intronic and intergenic regions compared to capturing polyadenylated RNA, which mostly contains mature protein coding transcripts. 6% 5% 4% % % % % Total RNA RNA-Seq Total RNA rrna-depleted in silico Lab Lab Lab KAPA RiboErase RNA-Seq KAPA mrna-seq Kit FIGURE Comparison of mrna capture and RiboErase sequencing data chr p. mrna Capture 7,74, bp [ - 5] Coverage p. p. 7,75, bp p. p. p. q q. 7,76, bp q. q. q4. q5 q. 5,48 bp 7,77, bp [ - 5] Junctions Reads [ - 4] [ - 4] RiboErase Coverage Junctions Reads GC Coverage RefSeq genes PHB SCARNA PHB q. q. q. 7,78, bp q q. q. q4. q4. 7,79, bp q4. q4. Libraries prepared using ng of Universal Human Reference RNA (UHR) with KAPA Stranded RNA-Seq Kit with RiboErase provides additional expression information of important RNA species such as noncoding RNA, which are absent with traditional mrna capture methods. KAPA RiboErase detects transcripts of the 7 bp small Cajal body-specific RNA (scarna - as outlined in red) located in an intron of the prohibitin (PHB) gene. The non-coding RNA is predicted to guide the pseudouridylation of residue 46 in U5 spliceosomal RNA.

5 INDUSTRY-LEADING, CONSISTENT rrna DEPLETION KAPA RiboErase constantly outperforms Illumina Ribo-Zero Gold, with only trace quantities of rrna remaining post-depletion. Results are consistent across multiple inputs and replicates. FIGURE 4 KAPA RiboErase delivers consistently better rrna removal compared to Illumina Ribo-Zero Gold 8S rrna Carryover (%) KAPA Illumina Various amounts of UHR RNA were depleted of rrna in triplicate using both the KAPA Stranded RNA-Seq Kit with RiboErase workflow and the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold protocol. Residual rrna in the samples was measured using qpcr, targeting the 8S rrna and GAPDH mrna transcripts. The results were plotted on a logarithmic scale. In all samples, KAPA RiboErase significantly outperformed the Illumina protocol in terms of 8S rrna depletion ng 5 ng ng ng Total RNA Input VERSATILE AND EFFECTIVE, rrna DEPLETION FROM VARIOUS SPECIES KAPA RiboErase has been designed to deplete rrna from human, mouse, rat, and related species such as primates. Intact or degraded total RNA extracted from a wide variety of sample types can be used, including formalin-fixed, paraffin-embedded (FFPE), to make whole transcriptome libraries with the KAPA Stranded RNA-Seq Kit. FIGURE 5 Reproducible depletion of rrna from various sample types and amounts % Residual rrna 5% 4% % % KAPA RiboErase effectively depletes rrna from a wide variety of samples, including non-human primate and mouse. Depletion is effective with various quality and input amounts of RNA. Residual rrna levels are represented as percent of the total mapped reads. % %.5 UHR.6 HBR.5 ng.4.4 ng. 5 ng. ng ng 5 ng Degraded RNA FFPE HBR RIN.6 Human Rhesus Macaque Mouse

6 4 ACHIEVE HIGHLY REPRODUCIBLE SEQUENCING RESULTS To assess the performance of the KAPA Stranded RNA-Seq Kit with RiboErase, libraries were prepared with varying input amounts of Universal Human Reference RNA (UHR), with ERCC RNA Spike-In Control Mixes at three different test sites, and sequenced using an Illumina HiSeq 5 to a minimum of M reads per library. Gene expression levels were determined as RPKM, and used to assess the quality and reproducibility of KAPA Stranded RNA-Seq Kit with RiboErase for whole transcriptome sequencing. FIGURE 7A Hierarchically clustered heatmap showing the correlations of gene expression levels between different laboratories and RNA input amounts A All correlations were greater than.9, with technical replicates produced in the same lab exceeding.98. Generally, all libraries from a single lab, including those produced from different input amounts, clustered together. The data indicates that the KAPA Stranded RNA-Seq Kit with RiboErase delivers robust, reproducible, and unbiased gene expression data from a wide range of input amounts. ng - S ng - S ng - S4 ng - S ng ng - S ng - S Lab ng - S ng ng - S Lab ng - S ng - S ng Lab. Lab ng.99 ng - S ng - S.984 ng - S Lab ng.976 ng - S.968 ng - S ng - S B Comparison of three independent libraries constructed in Lab using replicates of ng UHR RNA show a very high correlation (R >.978). Similarly, high correlations of gene expression levels were observed between data sets derived from a range of input amounts..96 ng Lab.95 ng - S ng - S4.944 ng - S.96 ng - S FIGURE 7B Correlation of gene expression levels between samples produced in a single lab.986 RR==.986 ng ng - S - S - S ng ng - S ngng 5 ng 5 ng.946 R R= =.946 ng ng - S - S.997 RR==.997 ng ng - S - S RR== R R= = ng ng - SS ng ng - SS ng ng.9788 RR==.9788 ngng ng ng

7 FIGURE 8 Consistent depletion of rrna in three different laboratories Residual rrna (%) Highly efficient removal of rrna from all UHR samples in three labs is evident by the consistently low levels of residual rrna after sequencing, represented here as percentage of total mapped reads and compared to the input RNA sample Input RNA S S S S S S S S4 S S ng ng ng ng ng ng Lab Lab Lab FIGURE 9 Consistently even coverage across transcripts Coverage (Normalized) Lab - UHR ng Lab - UHR ng - S Lab - UHR ng - S Lab - UHR ng - S Lab - UHR ng - S4 Lab - UHR Overlaid plots comparing the normalized depth of coverage averaged across highly expressed transcripts in libraries prepared with various UHR inputs Normalized Position Along Transcript (5 to ) FIGURE Highly sensitive expression profiling analysis using ERCC Spike-In Mix and A Log (RPKM Mix : Mix) Fitted Line Pass Filter Filtered R = Expected Log (expected fold-change) B Log (RPKM Mix : Mix) Fitted Line Pass Filter Filtered R = Expected Log (expected fold-change) Libraries were prepared from total RNA supplemented with ERCC RNA Spike-In Mix or, allowing analysis of the fold-change response between two samples. A Comparison of two technically replicated ng UHR RNA samples. B Comparison of ng UHR and ng Human Brain Reference (HBR). KAPA Stranded RNA-Seq Kit with RiboErase allows the sensitive detection of very small but significant gene expression changes.

8 CONCLUSIONS The KAPA Stranded RNA-Seq Kit with RiboErase offers a high-quality, comprehensive solution for transcriptome sequencing from ng µg of total RNA from human, mouse, or rat samples. By utilizing a targeted enzymatic method for depletion, our workflow enables superior reduction of ribosomal RNA (up to 99.98%) and a more complete representation of the transcriptome, including precursor mrnas and long non-coding RNAs. These benefits have been measured in comparison to both poly-a selection and other bead-based ribosomal RNA methods. Along with improved sequence performance, the KAPA Stranded RNA-Seq Kit with RiboErase has been proven to be highly robust and reproducible across varying input amounts and independent test sites. Through the use of evolved enzymes including the KAPA HiFi DNA polymerase, high-quality reagents and an optimized with bead workflow, the kit has been designed to be automation-friendly, and has demonstrated improved coverage of difficult transcripts, including those that are GC rich and in low abundance.

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