Trio RNA-Seq offers a best-in-class, whole transcriptomics solution for low input, liquid biopsy, FFPE and other challenging samples. Designed for robust and reliable RNA-Seq library preparation, Trio RNA-Seq can help you capture complete biology from virtually any sample. Trio RNA-Seq integrates the proven technology from the highly published Ovation® RNA-Seq System V2 to provide a complete workflow from total RNA to library.
Trio RNA-Seq combines three powerful technologies:
- Single Primer Isothermal Amplification (SPIA) enabling access to limited and degraded samples.
- Enzymatic fragmentation and DimerFree library construction allowing efficient and robust library preparation.
- AnyDeplete customizable transcript depletion after library construction maximizing informative sequencing reads from whole transcriptome data.
This unique workflow provides whole transcriptome libraries with no adaptor dimers and integrated transcript depletion enabling increased detection and more uniform coverage of transcripts.
Trio RNA-Seq is offered with AnyDeplete probes targeting Human rRNA (Part no. 0506). AnyDeplete probe sets can be customized to any transcript from any organism. For custom probe sets, contact your Account Executive or request a quote on our website.
Consistent performance from wide input range
- High quality data from as low as 500 pg
- Strong correlation seen across input range
Unbiased transcript coverage
- Complete 5' to 3' transcript coverage
- Comprehensive transcript analysis
Excellent results from FFPE and/or degraded samples
- Highly reproducible results from low quality RNA
- Access to previously inaccessible biologically relevant samples
- RNA sequencing (RNA-Seq)
- Whole transcriptome profiling
- Gene Expression
- Transcript discovery
- Viral detection
|Compatible Platform||Illumina HiSeq, MiSeq, NextSeq|
|Starting Material||Total RNA|
|Input Range||500 pg - 50 ng|
Trio RNA-Seq System provides all necessary buffers, primers, enzymes and depletion probes for cDNA synthesis, library construction, and targeted depletion. A control RNA (K562 total RNA) is provided to enable testing of the workflow. Nuclease-free water, Agencourt Beads and EvaGreen for the optional PCR optimization step are not provided.
What is the concentration is the control RNA?
The control RNA (K562 total RNA) is provided at a concentration of 1 μg / μL.
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 Tapestation 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?
Trio 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.
Can this system be used without AnyDeplete targeted depletion?
Yes. Please contact NuGEN technical support at email@example.com for information regarding the no-AnyDeplete workflow.
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.
How much total RNA do I need for library generation?
Trio RNA-Seq can be used with purified total RNA in the range of 500 pg to 50 ng. Input amounts outside this range may produce unsatisfactory and variable results.
Do I need to perform an rRNA depletion or poly(A) enrichment step before processing samples with Trio RNA-Seq?
rRNA depletion or Poly(A) enrichment is not required. The input range of 500 pg to 50 ng refers to total RNA.
Can I use poly(A)+ RNA as an alternative to total RNA?
The RNA-Seq System has not been tested for use with poly(A) RNA.
Do I need to use high-quality total RNA?
Trio RNA-Seq is designed to work with purified total RNA. When using purified total RNA, samples should be of high molecular weight with little or no evidence of degradation. 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 using total RNA inputs of 10–50 ng. With highly degraded samples, users may experience reduced sequencing metrics.
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.
Can I skip DNase treatment if I already incorporated DNase treatment during RNA isolation?
Residual amounts of genomic DNA can be amplified in final libraries and impact sequencing results. For this reason we recommend performing DNase treatment as a part of the Trio RNA-Seq workflow, even if samples have already been treated.
Can I use Trio RNA-Seq with RNA from any organism?
Trio RNA-Seq System has been designed for use with total RNA isolated from a broad range of different organisms. The system requires obtaining organism-specific probes to target specific transcripts for depletion. See Table 2 in the User Guide for a list of available probe sets, or contact NuGEN Technical Support at firstname.lastname@example.org for more information on designing custom primers.
We recommend a minimum batch size of 4 reactions for an 8 reaction kit, and 8 reactions for a 32- or 96- reaction kit. Smaller batch sizes may result in difficulty pipetting small volumes and lead to poor performance. In addition, this ensures sufficient reagent recoveries for the full number of reactions in the kit. Making master mixes for fewer than 4 or 8, respectively, samples at a time may affect reagent recovery volumes.
Does Trio RNA-Seq deplete ribosomal RNA?
Yes. Trio RNA-Seq features NuGEN's targeted depletion technology which can be customized to any transcript, any organism.
Can I modify the number of PCR amplification cycles recommended by Trio RNA-Seq when using different RNA input amounts?
Generally speaking, fewer PCR cycles will be needed when working with larger input amounts. See Table 12 of the User Guide for guidelines on the number of cycles to use.
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.
Are Trio RNA-Seq libraries stranded?
No, libraries prepared with Trio RNA-Seq are not stranded.
Where can I safely stop in the protocol?
It is safe to stop after SPIA Amplification, Adaptor Ligation Purification, Library Amplification I, Library Amplification II and Library Amplification II Purification. After SPIA Amplification and Library Amplification II, store reaction products only at 4° C overnight.
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.II. 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 Trio RNA-Seq adaptors add to the library?
The adaptors add 122 bp to the library.
What is the expected library size?
Trio RNA-Seq libraries generated with high-quality human total RNA are 300 bp on average.
What sequencers are compatible with your libraries?
Trio RNA-Seq libraries are compatible with Illumina sequencing platforms.
What kind of sequencing primers can I use with your library?
Trio RNA-Seq is designed for use with the standard Illumina sequencing primers for both single end and paired-end sequencing applications. Illumina sequencing primers are used for multiplex sequencing.
Can Trio RNA-Seq be used with paired-end sequencing?
Yes, Trio RNA-Seq can be used for both single end and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The expected distances between the 5’-most and 3’-most coordinates of paired-end reads will depend on the average fragment size of the insert pool.
What percentage of rRNA reads can I expect 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).
Please follow manufacturer’s recommendations for library QC, quantitation, balancing and loading of the amplified library on the sequencer. For eperiments using the Ovation Rapid DR Multiple Systems 1–8 and 9–16, also see Appendi A. of the Ovation Rapid Library Systems User Guide.
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.
Trio RNA-Seq, Human rRNA
8, 32, 96, Automation
Trio RNA-Seq with custom AnyDeplete probe set
Contact your Account Executive
For research use only. Not for use in diagnostic procedures.