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Targeted RNA sequencing

Targeted RNA sequencing with exome, custom, or predesigned hyb capture panels has high mapping, on-target percentage, and low duplication rates. Targeted RNA sequencing decreases intronic and intergenic reads to focus your data on exonic sequences.

xGen™ NGS—made for targeted RNA sequencing.

Overview

  • Fast and reliable—prepare RNA-seq libraries in 3.5 hours
  • Provides comprehensive data—for mapping, on-target, and transcript coverage
  • Compatible with a range of input types and quantities—prepare RNA-seq libraries from low-input and/or low-quality RNA samples using xGen Broad-Range RNA Prep Kit
  • Consistent libraries—minimal adapter dimers, so adapter titration is not required
  • Variety of indexing options available—for a total of 1536 Unique Dual Index (UDI) primer pairs
  • Flexible and customizable—combine with xGen Hyb Capture Panels for targeted RNA-seq
  • Automation-friendly RNA-seq workflow

What is targeted RNA sequencing?

Targeted RNA-seq is a cost-effective tool for deep sequencing of specified regions of interest within the transcriptome. This approach provides deep sequencing of targeted regions while omitting the undesired regions that often result in a disproportionate number of sequencing reads not relevant to the research study.

Reliable, time- and cost-effective approach to targeted RNA-seq

The xGen™ Broad-Range Library Prep Kit supports a wide input range, including low-quality RNA samples. This stranded RNA-seq workflow for low-quality and/or low-input RNA samples utilizes Adaptase™ technology to produce libraries following first-stand cDNA synthesis (Refer to xGen Broad-Range Library workflow). Generate RNA-seq libraries using Formalin Fixed Paraffin Embedded (FFPE) RNA research samples and for samples containing a wide range of input quantities and a variety of FFPE qualities. It is a recommendation for samples to have a RIN > 2 or DV200 > 30. Easily combine RNA library prep workflows with the xGen UDI Primer Plate for indexing.

Pair the xGen Broad-Range Library Prep Kit with the xGen Exome Hyb Panel v2 for a higher expression profiling efficiency, which yields a higher percentage of coding bases and a lower percentage of intronic bases than whole transcriptome data.

The xGen RNA Library Prep Kit offers a fast NGS transcriptomic research workflow. Produce RNA-seq libraries using tissue samples, blood, or high-quality RNA samples with a RIN of 7–10. This RNA-seq workflow utilizes the Adaptase technology to produce libraries following first-strand cDNA synthesis. (Refer to xGen RNA Library workflow). Easily combine RNA library prep workflows with xGen UDI Primer Plate for indexing.

Use an upstream poly(A) selection module of your choice and/or start with total RNA to use hybridization panels, e.g., xGen Custom Hyb Cap Panel designed to target and enrich genetic regions of interest.

Method data

Libraries were prepared using the xGen Broad-Range RNA Library Prep Kit using a degraded FFPE sample. The libraries were used as a single-plex or in a 4-plex hybridization capture with the predesigned xGen Exome Hyb Panel v2. An efficient library prep and hybridization capture workflow results in high mapping, on-target percentages, and low duplication rates. Captured mRNA libraries result in an increased number of exonic sequencing reads due to the decreased amount of intergenic and intronic reads when compared to the transcriptome libraries. This translates to sequencing cost efficiency due to reducing the number of unusable reads. All libraries had a removal of >99% of rRNA bases, indicating that ribodepletion is unnecessary for hybridization captured libraries (Figure 1).

Figure 1. Target enrichment metrics. Extracted FFPE RNA was used to prepare xGen Broad-Range RNA libraries from 10 ng of rRNA-depleted RNA (n = 3) or 50 ng of total RNA (n = 7). Hybridization capture using the xGen Exome Hyb Panel v2 was performed on total RNA libraries as single-plex (n = 3) or in a 4-plex hybridization (n = 1). All libraries were sequenced on a NextSeq® (Illumina®) 300 cycle kit and subsampled to 40 million reads per sample. (A) The data shows comparable mapping rates and duplication rates, and higher exonic reads and lower intronic reads for hybridization captures samples. (B)The feature bases chart shows that hybridization capture results in a higher percent of coding bases sequenced, and fewer intronic bases than transcriptome libraries (values within bars represent means of the replicates).

xGen RNA Library Prep Kit with xGen Custom Hyb Panels

Figure 2. xGen Custom Hyb Panels provide high quality sequencing results according to NGS performance metrics. The xGen RNA Library Prep Kit was used to generated sequencing libraries with 100 ng or 500 ng of total UHR RNA followed by hybridization capture using one of two xGen Custom Hyb Capture Panels (Pan-Cancer Genomic Panel for RNA or DNA applications and Pan-Cancer Transcript Panel for RNA applications), and then subsampled to 2 million reads per sample. Sequences were processed and aligned using STAR [1] and the hg38 reference genome [2]. This workflow resulted in (A) a mapping rate of >78%, (B) on-target rate of >98%, and (C) and correct strandedness of >99.8%. ( D) Sequencing shows the number of unique molecules scales as expected based on the RNA input amount into library prep (Picard) [3]. Values within the bar chart represent the mean of three libraries multiplexed in one hybridization capture.

References

  1. Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15-21.
  2. Kent WJ, Sugnet CW, Furey TS, et al. The human genome browser at UCSC. Genome Res. 2002;12(6):996-1006.
  3. “Picard Toolkit.”. Broad Institute, GitHub repository: Broad Institute; 2019. https://broadinstitute.github.io/picard/

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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