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Revelo™ RNA-Seq High Sensitivity library preparation kit

Whole transcriptome solution optimized for the detection and characterization of pathogen (rare and low abundance) transcripts from degraded samples
Overview
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Tab 01 / Overview
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High Sensitivity RNA-Seq library preparation kit

Optimized for degraded, low input samples

RNA-Seq library preparation from degraded or mixed samples, where detection of low abundance sequences is critical, poses a serious challenge to many researchers including high background.

 

Selection guide

Revelo RNA-Seq High Sensitivity is a whole transcriptome solution offering end-to-end processing of human or mouse total RNA samples in 6.5 hrs. Optimized for the detection and characterization of rare and low abundance transcripts from degraded samples with inputs as low as 250 pg, this kit is suitable for FFPE samples, nasal swabs, and blood samples.

Revelo RNA-Seq High Sensitivity library preparation kit offers several unique benefits:

  • SPIABoost™ technology for improved human rRNA and globin mRNA or mouse rRNA suppression to maximize informative sequencing reads
  • Enzymatic fragmentation for efficient sample preparation
  • Unique dual indexed (UDI) Adaptors for increased multiplexing and detection of index hopping
  • Eliminate adaptor dimers without adaptor titration regardless of sample input using DimerFree® technology
  • NuQuant® library quantification eliminates the need for expensive, time-consuming quantification methods such as qPCR. Using a simple fluorescent measurement with a Qubit or any standard plate reader, libraries can be quantified within minutes
  • Ready-to-go automation scripts available with the DreamPrep™ NGS workstation

Applications

  • RNA sequencing (RNA-Seq)
  • Whole transcriptome profiling
  • Gene Expression
  • Pathogen discovery
  • Viral detection




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Tab 02 / Highlights
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Sensitive detection of SARS-CoV-2 from highly degraded nasal swab samples

Revelo RNA-Seq High Sensitivity features high-sensitivity detection of viral sequences from as low as ~100 copies at a low read depth of 1M reads/sample. This exceptional sensitivity leads to the reduction in sequencing costs while still maintaining reliable high-quality results from samples of varying quality.

The quality of total RNA extracted from nasal swabs for these six samples was highly degraded with a majority of each sample composed of smaller RNA fragments (RIN values <3.0)

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Successfully process degraded RNA from nasal swab samples

  • BioAnalyzer trace indicating the sample quality for total RNA samples extracted from nasal swabs

 

Increased viral genome coverage

  • ≥5 more coverage compared to a competitor kit at 100 and 1,000 copies
  • Increased detection sensitivity enables access to high-quality data from as little as 100 viral copies and uncovers previously inaccessible information from as little as 500 pg total RNA input. ≥1 x and ≥10 x showed a similar trend of increased viral genome coverage

 

Exceptional sensitivity

  • Generate high quality libraries from total RNA extracted from nasal swab samples of varying quality and viral copies. RNA samples with RIN values as low as 2.20 and Ct values for SARS-CoV-2 detection ranging from 19 - 32 were successfully processed

 

Reduce the noise with SPIABoost technology

Revelo RNA-Seq High Sensitivity suppresses unwanted human rRNA and globin transcripts or mouse rRNA using proprietary SPIABoost technology to maximize informative sequencing reads.

Competitor T libraries show 8-10% rRNA reads in the final libraries, compared to <1% in Revelo-Seq. This leads to increased informative reads, reduced sequencing costs, and simplified data analysis.

Unbiased human transcript coverage with Revelo RNA-Seq High Sensitivity

RNA-Seq is a powerful tool for gene expression studies.

An even 5' - 3' human transcript coverage as seen with Revelo RNA-Seq High Sensitivity libraries enables comprehensive transcript analysis.

 

Streamlined sample-to-sequencer workflow with integrated library quantification in ~6.5 hrs

NuQuant library quantification eliminates the need for expensive, time-consuming quantification methods such as qPCR. Using a simple fluorescent measurement with a Qubit or any standard plate reader, libraries can be quantified within minutes.

Ready-to-go automation scripts available with the DreamPrep™ NGS workstation.

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Ready-to-go automation scripts available

  • Automation-friendly kit that has also been implemented on Tecan’s DreamPrep NGS workstation to offer a complete, automated solution for walkaway processing, helping to increase throughput and efficiency

 

Libraries can be quantified in minutes

  • Nuquant eliminates the need for expensive, time-consuming quantification methods such as qPCR

 




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Input Range

Input Range

250 pg - 10 ng

Starting Material

Starting Material

Total RNA

Suppression

Suppression

• Human nuclear rRNA, mitochondrial rRNA and adult globin transcripts
• Mouse nuclear rRNA and mitochondrial rRNA

Multiplexing

Multiplexing

Up to 96 single index and 96 UDI

Automation

Automation

DreamPrep NGS (Fluent 780 workstation). Other automation platforms available.

Sequencing compatible platform(s)

Sequencing compatible platform(s)

Illumina HiSeq, MiSeq, NextSeq, MiniSeq, NovaSeq




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Tab 04 / Faq
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FAQ

Getting started

What materials are provided with Revelo RNA-Seq High Sensitivity?

The Revelo RNA-Seq High Sensitivity kit provides all necessary buffers, primers and enzymes for cDNA synthesis, human or mouse suppression, SPIA amplification, library construction and NuQuant. SPRI purification beads and EvaGreen are not included.

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, a magnetic plate for 0.2 mL tubes, strips, or plates and a spectrophotometer or fluorometer. An Agilent Bioanalyzer or Fragment Analyzer may also be useful for optional analytical tests. A comprehensive list of required and recommended equipment can be found in Section II.B.

Can this system be used with other library preparation workflows?

Revelo RNA-Seq High Sensitivity is an end-to-end solution designed to generate libraries for Illumina sequencing starting from total RNA and has not been tested with alternative library preparation systems.

Input recommendations

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

  • Norgen Biotek Total RNA Purification Kit
  • Zymo Research Quick-RNA™ Kits
  • Arcturus PicoPure® RNA Isolation Kit
  • Ambion PureLink® RNA Mini Kit
  • Qiagen RNeasy Kits

For FFPE RNA isolation, we recommend a kit designed for FFPE samples, including:

  • Norgen Biotek FFPE RNA Purification Kit
  • Zymo Research Quick-RNA™ FFPE Kit
  • Arcturus® Paradise® PLUS FFPE RNA Isolation Kit
  • PureLink™ FFPE RNA Isolation Kit
  • Qiagen RNeasy FFPE Kit

Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method. Isolation methods that capture RNAs shorter than 200 nt may result in increased 5S and 5.8S rRNA. Do not use carrier RNA during isolation.

Can I use TRIzol® or other phenol-chloroform based extractions for RNA isolation?

We do not recommend the use of TRIzol or similar methods as any carryover of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.

Can I use carrier RNA during RNA isolation?

We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Tecan NGS Technical Support for more information.

Can I use Revelo RNA-Seq High Sensitivity with RNA from any organism?

Revelo RNA-Seq High Sensitivity has been designed for use with total RNA inputs from human or mouse samples.

Do I need to use high-quality total RNA?

Revelo RNA-Seq High Sensitivity is designed for whole transcriptome RNA-Seq and will work well with high-quality total RNA. The kit has also been shown to be compatible with degraded samples such as RNA extracted from FFPE or nasal swabs. Contact Tecan NGS Technical Support for more information.

Do you recommend DNase treatment of purified total RNA samples?

Yes. When using purified total RNA samples, contaminating genomic DNA may amplify during the process. For this reason we recommend DNase treatment during RNA purification.

General workflow

How much extra reagent is recommended when preparing the master mixes at each step?

A minimum amount of overage should be used in master mixes to ensure the full nominal number of reactions in the kit. The amount of overage needed depends on sample batch size, pipetting accuracy, and viscosity of reagents. We have found that 10% extra volume is sufficient at most steps. When preparing master mixes with particularly viscous components, including Fragmentation and End Repair, and Adaptor Ligation, 12-15% extra volume is recommended.

My input RNA samples are already fragmented (e.g. RNA derived from plasma, FFPE, nasal swabs, etc.). Can I skip the fragmentation and end repair step?

The fragmentation and end repair step is required in the Revelo RNA-Seq High Sensitivity workflow. This kit has been demonstrated to work with moderately degraded samples.

Can I combine the barcoded libraries prior to the PCR amplification step?

This is not recommended. The stoichiometry of barcoded libraries may be adversely affected by this modification to the workflow. We suggest that the libraries be amplified and quantified independently before being pooled for use on the sequencer.

Where can I safely stop in the protocol?

Samples can be placed in short-term storage at –20 °C after any of the bead purification steps.

How long can I store Revelo RNA-Seq High Sensitivity libraries at –20 °C?

Libraries are stable at –20 °C for at least 4 months. Libraries must be protected from light.

SPRI bead purifications

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP beads in our kits and observe no difference in performance between products.

What magnetic separation devices do you recommend for the SPRI bead purifications?

Due to the large number of commercially available magnets, we do not have a comprehensive list of compatible products. However, many magnets are compatible. As long as the magnet is strong enough to clear the solution of magnetic beads, it can be applied to the system. We have the following guidelines for selecting a magnetic separation device:

  1. Use a magnet designed for 0.2 mL tubes (PCR tubes), tube strips, or plates. Compared to magnets that are designed for 1.5 mL tubes, these minimize loss that can occur when samples are transferred from one tube to another.
  2. Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
  3. Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the Tecan workflow. This is also helpful to gain familiarity with the purification workflow.

How can I ensure maximum recovery of sample from the SPRI bead purification?

  1. Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
  2. Ensure that the beads are fully resuspended in solution before adding to the sample.
  3. Always use fresh ethanol during the washing steps. When preparing the ethanol, measure out the ethanol and water separately to ensure the desired ethanol concentration is obtained.
  4. Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

Library quantification and qualification

How do I measure my final library yield?

We recommend using NuQuant to accurately quantify the final libraries for multiplex p.ooling using a Qubit or plate reader. The final library pool concentration should be determined using a qPCR-based method before loading onto an Illumina sequencer. Please refer to Section V. N. for guidelines on alternative library quantitative and qualitative assessments.

How many bases do Revelo RNA-Seq High Sensitivity adaptors add to the library?

The adaptors add 136 bp to the library.

Sequencing recommendations

What sequencers are compatible with your libraries?

Revelo RNA-Seq High Sensitivity libraries are compatible with Illumina sequencing platforms.

How much material should I load into the sequencer?

Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.

What kind of sequencing primers can I use with your libraries?

Revelo RNA-Seq High Sensitivity libraries are designed for use with the standard Illumina sequencing primers for both single-end and paired-end sequencing applications.

Can Revelo RNA-Seq High Sensitivity libraries be used with paired-end sequencing?

Yes. The libraries produced using this kit can be used for both single- and pairedend sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The workflow generates libraries with an average insert size of approximately 300 bases. Contact Tecan NGS Technical Support for additional information.

Do Revelo RNA-Seq High Sensitivity libraries have special data analysis requirements?

Once the data have been parsed according to sample, a specialized adaptor trimming command is required at the 5’ end of the reads. To do this we recommend using cutadapt (https://cutadapt.readthedocs.io/en/stable/) with -g ACTTTGTGTTTGA for single end (or -g ACTTTGTGTTTGA -G ACTTTGTGTTTGA for paired end). For data sequenced on an Illumina instrument using two-color chemistry (NextSeq, NovaSeq, MiniSeq), we recommend also adding --nextseq-trim=20 option in cutadapt.

Contact Tecan NGS Technical Support for more information.

Data analysis

What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?

Each barcode is a minimum edit distance of 3 from any other barcode. This means that a minimum of three edits (replacement, insertion, or deletion) must occur before one barcode becomes a different barcode. For further details on the barcode design strategy, please refer to Faircloth BC, Glenn TC (2012), Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels. PLoS ONE 7(8): e42543. doi:10.1371/journal.pone.0042543.




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Tab 05 / Literature
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Literature

Type
Title
No
Product Sheet
Pathogen detection and characterization with Revelo RNA-Seq
401800 V2.0
Application Note
401799 V3.0
User Guide
Revelo RNA-Seq
M01529 V4.0
Safety Data Sheet
 
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