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 Exome

The Twist RNA Exome, along with Twist RNA Library Prep and Twist Target Enrichment, provides a reliable tool for generating transcriptome sequencing data from RNA extracted from a variety of sources including FFPE. RNA Exome increases the signal while requiring fewer sequencing reads for the same number of genes detect. This enables the detection of low expressing targets that are critical for an accurate picture of the transcriptional state of the biological sample. Additionally, the RNA Exome’s exon-aware design approach leaves open the ability to detect isoforms and junctions that may otherwise get lost with typical design approaches. This complete sequencing solution can produce highly complex and uniform sequencing reads for RNA sequencing analysis of the protein coding sequences of the human transcriptome.

More Targets, Fewer Reads

Twist RNA Exome detects more targets with fewer reads over a range of RNA inputs with FFPE and universal human reference RNA. All libraries were prepared with the Twist RNA Library Prep.