Twist RNA Sequencing Workflow Solutions

Twist’s RNA sequencing workflows offer a complete NGS solution that produces uniform libraries for RNA sequencing. Twist offers targeted and whole transcriptome workflows that reduce time at the bench and integrate with our current set of NGS reagents, including our target enrichment kits. Each workflow delivers high quality libraries ready for sequencing from a wide range of inputs and sample types, including translation research samples.

Twist Custom RNA Panels can be designed and built to cover a wide range of panel sizes and transcript target regions with the exceptional and consistent performance. Utilizing our deep design experience, Twist has developed exon-aware design algorithms to target individual exons while not preferentially selecting any one exon-exon junction site. We’ve applied this design approach to the Twist RNA Exome which makes it a good tool for transcriptome analysis and fusion detection.

The end-to-end workflows achieve this by combining streamlined and effective library preparation kits, high quality unique dual indices (UDI), unique molecular identifiers, optimized target enrichment, and proprietary exon-aware panel design algorithm.

Targeted RNA Sequencing

• ​​Create custom panels to sequence only the RNA transcripts that matter 
• Combine Twist RNA Library Prep, Twist Hybridization and RNA target enrichment panels
• Enrich sensitivity for key targets even in low quality, degraded RNA from FFPE

RNA Exome & Targeted Sequencing

• ​​Exon-aware probe design for protein coding regions and unique splice sites
• Fewer reads per sample for more samples
• Targets 19708 genes and 63215 isoforms 
• Greater than 1.8 fold enrichment over whole transcriptome

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Whole Transcriptome Sequencing

• Measure expression levels of mRNA, lncRNA and other RNA species
• Prepare libraries from Total RNA extracted from fresh and FFPE samples
• Combine our Twist RNA Library Prep with Twist rRNA & Globin Depletion kit
• Perform RNA Library Preparation in less than 5 hours

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RNA Fusion Detection

• Enrich for known and novel fusions with the Twist Alliance CeGaT Fusion Panel
• Expert content design through collaboration with CeGaT
• Optimized complete workflow with Twist RNA Library Prep kit for sequencing ready library
• Leverage Twist core capture enrichment technology to maximize efficiency

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RNA-Seq Library Preparation Overview

Twist RNA library preparation offers workflow which was optimized to take a sample through to completed library in as little as 5 hours (left). The whole transcriptome libraries can be sequenced directly for comprehensive analysis. They may also be used as input into Twist RNA target enrichment workflow with our Target Enrichment panels for RNA Exome, RNA Fusion, or custom designed RNA enrichment panels which focus NGS coverage on coding regions enabling gene expression analysis, fusion detection, and identification of coding variants from a single assay (right).

RNA-Seq Library Preparation Workflow

Starting with total RNA as input from 1 ng up to 1 ug of input can be introduced into the workflow with or without the optional ribosomal RNA and Globin depletion module to remove unwanted RNA species. The RNA is fragmented and then reverse transcribed into cDNA followed by a single-step 2nd strand synthesis and A-tailing that saves time and procedure steps. Finally, libraries are constructed by ligating Twist high quality UMIs or Universal Adaptors (UDIs) followed by amplification.

RNA Fusion

Gene fusions often act as oncogenic driver mutations in a wide variety of cancer types. Their rapid and accurate detection can inform clinical actions and drive treatment decisions. The Twist Alliance CeGaT RNA Fusion Panel Kit is an RNA-based enrichment panel and offers an improved targeted and sensitive approach to investigate gene fusions than the conventional methods.

This panel includes 160 fusion genes associated with approximately 30 cancer types. The fusion genes were selected by CeGaT, a leading genetic diagnostic and sequencing company located in Europe. The design is not only for the detection of known gene fusions but is also used to identify novel gene fusions.

The panel is optimized and validated for use with the Twist RNA Library Prep Kit for a complete end-to-end workflow exceptional performance. High capture efficiency with the RNA-based enrichment increases the sensitivity of detection. This panel is ideal for screening oncology samples to inform treatment options or discover novel fusions for tumor classifications.*

Panel Design

• 160 fusion associated genes with 66 genes in the breakpoint design
• 7394 Probes
• Covers a variety of different cancer types

* Note: The Twist Alliance CeGaT RNA Fusion Panel – 3 MB is ISO-13485 certified.