Revelo RNA-Seq for pathogen detection

Product Details

High Sensitivity RNA-Seq library preparation kit

One-day sample to sequencer solution for pathogen detection

RNA-Seq library preparation from degraded or mixed samples, where detection of low abundance sequences is critical, poses a serious challenge to many researchers. Most commercially available RNA-Seq kits are inadequate to sensitively detect and characterize low-titer viral or other pathogenic agents from samples in high human background, such as nasal swab samples SARS-CoV-2 infected patients. These samples may have a wide range of background contamination based on the sample collection method, impacting the ability to detect low viral copy numbers with confidence.

The Revelo RNA-Seq kit is a whole transcriptome solution offering end-to-end processing of human total RNA samples to enable complete RNA library preparation – including library quantification – in 6.5 hrs. Revelo RNA-Seq is a whole transcriptome solution optimized for the detection and characterization of rare and low abundance transcripts from degraded samples. This library preparation kit is integrated with an array of novel technologies for end-to-end processing of degraded samples with inputs as low as 250 pg.

New SPIAboost™ technology for improved human rRNA depletion to maximize informative sequencing reads
Enzymatic fragmentation for efficient sample preparation
Unique dual indexed 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

Back to RNA-Seq

Product Highlights

Sensitive detection of SARS-CoV-2 from highly degraded nasal swab samples

Revelo RNA-Seq 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).

Enhance informative reads with the new SPIAboost technology

Revelo RNA-Seq effectively eliminates unwanted human ribosomal reads (<1% rRNA reads) using proprietary SPIAboost technology for an improved depletion. 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

RNA-Seq is a powerful tool for gene expression studies. An even 5' - 3' human transcript coverage as seen with Revelo RNA-Seq 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

Applications

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

Specifications

Specification	Description
Input Range	250 pg - 10 ng
Starting Material	Total RNA
Ribosomal depletion	Human rRNA
Multiplexing	Up to 96 single index and 96 UDI
Automation	DreamPrep NGS (Fluent 780 workstation). Other automation platforms available.
Sequencing compatible platform(s)	Illumina HiSeq, MiSeq, NextSeq, MiniSeq, NovaSeq

Resources

Guides and Protocols

User Guide: Revelo RNA-Seq

Literature

SDS

Safety Data Sheet: Revelo RNA-Seq

FAQs

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Getting started

What materials are provided with Revelo RNA-Seq?

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

What equipment is required or will be useful?

A comprehensive list of required and recommended equipment can be found in Section II. B in the userguide

Can this system be used with other library preparation workflows?

Revelo RNA-Seq 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.

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 carry over 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 Technical Support for more information.

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

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

Do I need to use high-quality total RNA?

The Revelo RNA-Seq kit 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 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, large amounts of 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 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 quantitated 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 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:

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.
Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the NuGEN 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?

Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
Ensure that the beads are fully resuspended in solution before adding to the sample.
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.
Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

Library quantification/qualification

How do I measure my final library yield?

We recommend using NuQuant to accurately quantify the final libraries for multiplex pooling 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 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 libraries are compatible with Illumina sequencing platforms.

How much material should I load into the sequencer?

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

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.

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

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

Ordering Information

Description	Part No.	Reaction Sizes
Revelo RNA-Seq, Human rRNA	30184147	8
Revelo RNA-Seq, Human rRNA	30184149	32
Revelo RNA-Seq, Human rRNA	30184151	96, Automation
Revelo RNA-Seq, UDI Human rRNA	30184204	96, Automation

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Revelo™ RNA-Seq library preparation kit