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    Trio RNA-Seq™ library preparation kit

    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.
    Overview
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    Tab 01 / Overview
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    Product News: Trio RNA-Seq is no longer available.

    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.

     

    Selection guide

    Sensitive detection of rare transcripts from challenging samples

    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.

     

    Benefits of Trio RNA-Seq library preparation kit:

    • RNA sequencing solution for liquid biopsy, FFPE, nasal swab and other challenging samples with low input.
    • Customize rRNA depletion after library construction maximizing informative sequencing reads from whole transcriptome data.
    • Enzymatic fragmentation and DimerFree library construction allowing efficient and robust library preparation.
    • Detect index hopping with UDI.
    • Input range 500 pg - 50 ng of total RNA.
    • Ideal for detection and characterization of COVID-19 and infectious disease agents.

    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.

    Applications

    • RNA sequencing (RNA-Seq)
    • Whole transcriptome profiling
    • Gene expression
    • Transcript discovery
    • Viral detection




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    Tab 02 / Highlights
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    Increased detection sensitivity

    • Trio RNA-Seq increases detection sensitivity from nasal swab samples and is the method of choice for IGATech. (Data has been generated and analyzed by IGATech in the framework of a research initiative funded by ARGO, Italy).
    • Recent, peer-reviewed publications have demonstrated the utility of Trio RNA-Seq in the detection of a novel coronavirus from patient samples as well as providing insight into the SARS-CoV-2 evolutionary history and virulence.
    Show more

    Viral detection from Nasal swab samples

    The chart shows detection of viral sequencing even in the presence of high bacterial background

    Trio RNA-Seq sensitivity in detecting SARS-CoV-2

    • The table shows the sensitivity of Trio RNA-seq in detecting SARS-CoV-2 from nasal swab samples with varying Ct values. Complete viral sequence coverage achieved with 100M reads for Ct<27, 50M reads for Ct<25 and 10M reads for Ct<22 highlighting the sensitivity of the kit.
    • The final library yield was consistent (with an average of 28 nM) across the different input amounts showing the robustness of the SPIA amplification combined with the library preparation, making it suitable for processing samples of widely varying inputs and viral load.

    Consistent performance from wide input range

    • High quality data from low input RNA samples, as low as 500 pg
    • Strong correlation seen across input range
    Show more

    Distribution of reads in libraries prepared with Trio RNA-Seq

    Correlation analysis of libraries prepared with Trio RNA-Seq

    Unbiased transcript coverage

    • Complete 5' to 3' transcript coverage
    • Comprehensive transcript analysis

    Transcript coverage across the entire transcript length

    Excellent results from FFPE and/or degraded samples

    • Highly reproducible results from low-quality RNA or rare transcripts
    • Access to previously inaccessible biologically relevant samples

    Distribution of reads in libraries prepared with Trio RNA-Seq, using inputs Human Liver FFPE RNA (RIN 3.6)




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    Tab 03 / Specifications
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    Specifications

    Specification

    Specification/Description

    Compatible Platform

    Compatible Platform

    Illumina HiSeq, MiSeq, NextSeq, NovaSeq

    Starting Material

    Starting Material

    Total RNA

    Input Range

    Input Range

    500 pg - 50 ng




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    Tab 04 / Faq
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    FAQ

    Getting started

    What materials are provided with Trio RNA-Seq?

    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 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?

    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 Tecan NGS technical support at techserv-gn@tecan.com for information regarding the no-AnyDeplete workflow.

    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.

    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?

    Trio RNA-Seq 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 data quality.

    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.

    Can I skip DNase treatment if I already incorporated DNase treatment during RNA isolation?

    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.

    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 Tecan NGS Technical Support at techserv-gn@tecan.com for more information on designing custom probes.

    General workflow

    Can I perform fewer than 4 or 8 reactions at a time?

    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 Tecan'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 quantified 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.

    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? Can I use an Agilent Bioanalyzer to evaluate the product?

    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.

    How many bases do the Trio RNA-Seq adaptors add to the library?

    The single-index adaptors add 122 bp to the library and the dual-index adaptors add 144 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- and paired-end 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.

    Sequencing recommendations

    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.

    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.

    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).




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    Tab 05 / Literature
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    Resources

    Type
    Title
    No
    User Guide
    Trio RNA-Seq
    M01440 V1
    Product Sheet
    401141 V1
    Poster
    Trio RNA-Seq Simple Robust Method For Viral Detection
     
    Sample Sheet
     
    Safety Data Sheet
    Trio RNA-Seq
     
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    For research use only. Not for use in diagnostic procedures.
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