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Unique RNA-Seq library prep combines three proprietary technologies to provide a streamlined solution for whole transcriptome RNA-Seq from low input and poor quality samples for rare transcript detection and unbiased pathogen discovery.
Looking for a whole transcriptome RNA-Seq solution similar to Trio RNA-Seq?
Check out Revelo™ RNA-Seq High Sensitivity for our new and improved RNA-Seq library preparation technology.
RNA sequencing library preparation from challenging or mixed samples, where detection of low abundance or rare transcripts is critical, is a serious challenge to many researchers. Standard RNA-Seq methods are not adequate for the generation of templates suitable for sequencing rare transcripts in mixed samples. Standard RNA-Seq kits are unable to detect viral pathogens such as SARS-CoV-2 or identify uniquely expressing genes in specific cell types from challenging tissues such as neurobiological samples.
Trio’s unique amplification technology (single primer isothermal amplification, SPIA) allows for the detection of low input amounts and the low abundance of pathogens like viruses. This unbiased amplification technique generates more molecules for library preparation compared to standard RNA-Seq workflows and preserves biological information from samples. SPIA can tolerate inhibitors from challenging sample sources enabling better amplification of your transcript of interest.
Trio RNA-Seq offers a best-in-class and highly published NGS library preparation system that combines SPIA with DimerFree adaptor ligation and AnyDeplete targeted transcript depletion, offering a streamlined solution for whole transcriptome RNA-Seq from low input and poor quality samples for rare transcript detection and unbiased pathogen discovery.
Trio low input RNA-Seq is offered with both AnyDeplete probes targeting human rRNA (Part No. 0506) and AnyDeplete probes targeting mouse rRNA (Part No. 0507). 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.
The chart shows detection of viral sequencing even in the presence of high bacterial background
Illumina HiSeq, MiSeq, NextSeq, NovaSeq
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.
The control RNA (K562 total RNA) is provided at a concentration of 1 μg / μL.
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.
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.
Yes. Please contact Tecan NGS technical support at firstname.lastname@example.org for information regarding the no-AnyDeplete workflow.
We recommend a column-based method, including:
For FFPE RNA isolation, we recommend a kit designed for FFPE samples, including:
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.
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.
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.
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.
rRNA depletion or Poly(A) enrichment is not required. The input range of 500 pg to 50 ng refers to total RNA.
Trio RNA-Seq has not been tested for use with poly(A) 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 data quality.
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.
We recommend performing DNase treatment as a part of the Trio RNA-Seq workflow, because residual amounts of genomic DNA can be amplified and impact sequencing results.
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 Tecan NGS Technical Support at email@example.com for more information on designing custom probes.
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.
Yes. Trio RNA-Seq features Tecan's targeted depletion technology which can be customized to any transcript, any organism.
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.
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.
No, libraries prepared with Trio RNA-Seq are not stranded.
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 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.
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:
Please refer to the User Guide section on Quantitative and Qualitative Assessment. We recommend using a qPCR based-method in combination with the Agilent Bioanalyzer or Fragment Analyzer for the most accurate quantification.
The single-index adaptors add 122 bp to the library and the dual-index adaptors add 144 bp to the library.
Trio RNA-Seq libraries generated with high-quality human total RNA are 300 bp on average.
Trio RNA-Seq libraries are compatible with Illumina sequencing platforms.
Trio RNA-Seq is designed for use with the standard Illumina sequencing primers for both single- and 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.
Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.
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.
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).
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For research use only. Not for use in diagnostic procedures.