Ovation® Universal RNA-Seq System
The Ovation® Universal RNA-Seq System is part of a group of products that provide an end-to-end solution for strand-specific RNA-Seq libraries ready for sequencing on Illumina platforms. These systems combine a simple, flexible workflow that accommodates a broad range of total RNA inputs from a number of sample types coupled with efficient depletion of uninformative transcripts including ribosomal RNA depletion using the Insert Dependent Adaptor Cleavage (AnyDeplete) technology. In addition to rRNA depletion, the AnyDeplete (formerly known as InDA-C) probes can be customized to target any high-abundant transcript (custom transcript depletion) from any organism leading to an increased dynamic range, reduced sequencing costs, and simplified data analysis. The table below provides a summary of catalog kits available for rRNA transcript depletion from model organisms. For custom transcript depletion contact NuGEN Technical Support (firstname.lastname@example.org).
|Organism||Depletion target||Product Name||Part No.|
|Human||ribosomal RNA + globin||Ovation® Human Blood RNA-Seq Multiplex System 1-8, 9-16||0337, 0338|
|Human||ribosomal RNA||Ovation® Human FFPE RNA-Seq Multiplex System 1-8, 9-16||0340, 0341|
|Mouse||ribosomal RNA + globin||Ovation® Mouse RNA-Seq System 1-16||0348|
|Rat||ribosomal RNA||Ovation® Rat RNA-Seq System 1-16||0349|
|Drosophila||ribosomal RNA||Ovation® Drosophila RNA-Seq System 1-16||0350|
|Arabidopsis||Chloroplast, Cytoplasmic and mitochondrial ribosomal RNA||Ovation® Arabidopsis RNA-Seq System 1-16||0351|
|Custom||Custom||Ovation® Universal RNA-Seq System 1-16||0343|
|Human||ribosomal RNA||Ovation® Universal RNA-Seq System 1-96 (Automation)||9102-A01|
Highly reproducible sequencing results
- Robust workflow provides consistency between operators
- High sample-to-sample reproducibility
Unrivaled sequencing data quality
- Detect more transcripts across a greater dynamic range
- Consistent coverage across transcripts
Efficient transcript depletion with AnyDeplete (formerly known as InDA-C)
- Ribosomal RNA or transcript depletion does not perturb transcriptome
- Increased sequencing efficiency and dynamic range
Customizable depletion of any transcript from any organism
- Simple, iterative design process
- Whole transcriptome profiling
- Gene expression
- RNA sequencing
- Transcript discovery
|Compatible Platforms||Illumina HiSeq, MiSeq, NextSeq, MiniSeq|
|Starting Material||Total RNA, poly(A) selected RNA|
|Input Amounts||10 ng – 100 ng|
- User Guide: Ovation Human FFPE RNA-Seq System
- User Guide: Ovation Human Blood RNA-Seq System
- User Guide: Ovation RNA-Seq System for Model Organisms
- User Guide: Ovation Universal RNA-Seq System
- Technical Report: Detection of Genomic DNA in Human RNA Samples for RNA-Seq
- Technical Report: A Comparison on Two Sample Preparation Methods for Strand-specific RNA-Seq Using Human Whole Blood
The Ovation Universal RNA-Seq System Core Kit provides all necessary buffers, primers, enzymes and SPRI purification beads, as well as nuclease-free water for purification elution steps. AnyDeplete (formerly known as InDA-C) probes are available separately or in a bundle with the Core Kit. For custom designs the researcher will provide AnyDeplete primers used for targeted transcript depletion.
What equipment is required or will be useful?
Required equipment includes a microcentrifuge; pipettes; vorteer; a thermal cycler; a magnetic plate for 0.2 mL tubes, strips, or plates; and a spectrophotometer or fluorometer. An Agilent Bioanalyzer or Tapestation may also be useful for optional analytical tests. A comprehensive list of required and recommended equipment can be found in Section II.B of the User Guide.
Can this system be used with other library preparation workflows?
The Ovation Universal RNA-Seq System 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.
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 etractions 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 the Ovation Universal RNA-Seq System with RNA from any organism?
The Ovation Universal RNA-Seq System has been designed for use with a broad range of different organisms. The system requires obtaining organism-specific primers to target specific transcripts for depletion. See Table 2 in the User Guide for a list of available probesets, or section IV.C for more information on designing custom primers.
Do I need to use high-quality total RNA?
While it is impossible to guarantee the highest levels of performance when using RNA of lower quality, this system should allow the successful analysis of a wide range of samples. For FFPE or degraded samples, we recommend increasing the amount of total RNA input to 100-250 ng. With highly degraded samples, users may eperience lower yields and may encounter affected sequencing metrics.
Do I need to perform an rRNA depletion, poly(A) enrichment step, or globin depletion before processing total RNA samples with the Ovation Universal RNA-Seq System?
No. The system is designed to use total RNA as input. rRNA depletion or poly(A) enrichment are not necessary. rRNA, as well as other transcript types, can be targeted for depletion using the AnyDeplete (formerly known as InDA-C) technology embedded in the workflow.
How much total RNA do I need for library generation?
We recommend 10-100 ng high quality RNA, 100-250 ng degraded or FFPE RNA, or 100-500 ng whole blood RNA as input into the kit. Please refer to section III.A of the User Guide for a discussion of RNA input requirements.
Can contaminating genomic DNA interfere with the Ovation Universal RNA-Seq System performance?
Yes, contaminating genomic DNA may be incorporated into libraries. For this reason we recommend DNase treatment during RNA purification. For an eplanation of DNase requirements see section III.A, step 4 of the User Guide.
No. The cDNA is generated with random and poly(T) primers, but no SPIA-based amplification is used.
Is it necessary to fragment my cDNA prior to End Repair and Adaptor Ligation?
No, fragmentation is not required with this kit and can be omitted with little or no impact on genes detected, FPKM concordance, or differential epression concordance. With some samples NuGEN has observed a modest elevation in the percentage of sequencing reads mapping to rRNA when using AnyDeplete (formerly known as InDA-C) primers targeting these transcripts. In addition, omitting Covaris fragmentation leads to a small increase in reads mapping to the 5’ end of transcripts.
Does NuGEN provide reagents for performing the fragmentation step of the protocol?
NuGEN does not provide the reagents used in the fragmentation steps. Please see section V.E. in the User Guide for recommendations for fragmentation with the Covaris instrument.
I don’t have access to a Covaris instrument. Can I use alternative fragmentation methods?
We have evaluated only Covaris fragmented DNA during the development of these systems. Other means of fragmentation, such as sonication or enzymatic fragmentation, may be suitable as long as the method generates a tight size distribution of cDNA fragments with a median size of 200-400 bp.
How does your protocol enable strand retention?
The Ovation Universal RNA-Seq System utilizes targeted degradation of an incorporated modified nucleotide to ensure library inserts all carry the same directionality.
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 second strand synthesis, after cDNA fragmentation or after any of the bead purification steps.
RNAClean P beads are certified to be RNase and DNase free. We have tested both RNAClean P and AMPure P 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 maimum 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 maimum binding of the samples to the beads.
Please refer to section V.O of the User Guide for guidelines on quantitative and qualitative assessment. We recommend using a qPCR based-method in combination with the Agilent Bioanalyzer or Tapestation for the most accurate quantification.
How many bases do the Ovation Universal RNA-Seq System adaptors add to the library?
The adaptors add 122 bp to the library.
Ovation Universal RNA-Seq Systems 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?
The Ovation Universal RNA-Seq System is designed for use with the standard Illumina sequencing primers for both single-end and paired-end sequencing applications.
Can the Ovation Universal RNA-Seq System be used with paired-end sequencing?
Yes. The libraries produced using this kit can be used for both single-end and paired-end sequencing. Special consideration should be given to the epected insert size in the paired-end assay. The workflow generates libraries with an average insert size that is dependent on the fragmentation conditions used.
Yes. Strand-specific reads can be processed and mapped to reference sequences using the same methods used for other RNA-Seq libraries. Note that in libraries generated by the Ovation Universal RNA-Seq System, the forward read corresponds to the sense strand.
What percentage of rRNA reads can I epect in my data?
The number of rRNA reads present in the sequencing results is dependent on the abundance of rRNA transcripts in the starting material. For a sample containing 10% mRNA and 90% rRNA, a 90% depletion of rRNA transcripts results in a sample containing 53% mRNA and 47% rRNA (i.e. 10% and 9% of the original pool of RNA, respectively).
Ovation® Human Blood RNA-Seq Multiplex System 1-8
Ovation® Human Blood RNA-Seq Multiplex System 9-16
Ovation® Human FFPE RNA-Seq Multiplex System 1-8
Ovation® Human FFPE RNA-Seq Multiplex System 9-16
Ovation® Mouse RNA-Seq System 1-16
Ovation® Rat RNA-Seq System 1-16
Ovation® Drosophila RNA-Seq System 1-16
Ovation® Arabidopsis RNA-Seq System 1-16
Ovation® Universal RNA-Seq System 1-16
Ovation® Universal RNA-Seq System 1-96 (Automation)
For research use only. Not for use in diagnostic procedures.