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Robust solution for stranded whole transcriptome RNA-seq library prep for any sample type and RNA quality
We have recently introduced Universal Plus Total RNA-Seq with NuQuant for whole transcriptome RNA-Seq without time-consuming mechanical fragmentation.
Whole transcriptome RNA sequencing (RNA-Seq) is data-rich and enables the collection of the full transcriptome for analysis, unlike conventional technologies such as qPCR and microarrays that are limited to predefined transcripts. RNA-Seq enables researchers to investigate both known and novel transcripts allowing the study of regulatory regions, global expression levels, detection of exons and introns, and examination of splicing patterns.
The Universal RNA-Seq library preparation kit integrates a host of innovative technologies to provide an efficient and cost-effective workflow for end-to-end solution for strand-specific RNA-Seq libraries. This RNA-Seq library preparation kit is integrated with NuQuant, a proprietary library quantification method for efficient and accurate quantitation of Next-Generation Sequencing (NGS) libraries. NuQuant enables RNA-Seq libraries to be quantified and pooled in a single day.
The Universal RNA-Seq library preparation kit supports a broad range of RNA inputs including degraded FFPE samples and maximizes sequencing results by eliminating uninformative reads with our customizable rRNA depletion technology, AnyDeplete. Unlike other depletion methods, transcript depletion with AnyDeplete occurs after library construction, meaning there is no manipulation of input RNA and no bias in the final library. AnyDeplete is completely customizable to remove any unwanted sequence in your library, providing high quality, cost-effective data from any sample source.
Benefits of Universal RNA-Seq library preparation kit:
Duplicate samples were prepared from 20 ng of human whole blood RNA using the Ovation Whole Blood Solution, according to the product user guides. RNA samples were isolated using the Agencourt RNAdvance™ Blood kit on the Beckman ArrayPlex. Targets were hybridized to Affymetrix GeneChip®U133_plus_2.0 arrays. Results of linear regression analysis show a strong signal correlation of R = 0.992, with 58% present calls, demonstrating very high reproducibility across transcript abundance with only 20 ng of input total whole blood RNA.
AnyDeplete probe mixes are modular and available for a number of standard model organisms allowing flexible depletion of unwanted reads.
Depletion of highly-abundant transcripts from your library leads to increased dynamic range, reduced sequencing costs, and simplified data analysis.Show more
The AnyDeplete probe mix can be customized to include probes from our expanding list of custom designs, mix different designs to target multiple transcripts or organisms or design new probes specific for your species or experiment. For custom transcript depletion contact Tecan NGS Technical Support.
AnyDeplete is able to efficiently eliminate rRNA reads from the library significantly reducing the number of uninformative reads, providing more useful data and lower sequencing costs
AnyDeplete efficiently reduces the amount of unwanted rRNA reads from standard mouse and Drosophila samples and can be customized to target unwanted reads from any organism such as P. gingivalis
Available custom AnyDeplete probes for Universal RNA-Seq with NuQuant library prep kit
NuQuant library quantification is a proprietary method by which fluorescent labels are incorporated into the library molecules during the library preparation process.
Each library molecule has an equivalent number of labels incorporated, regardless of the size of the library fragment, resulting in direct measurement of the molar concentration using standard fluorometers.Show more
NGS library quantification with NuQuant is accurate, easy to use and is integrated with the Universal RNA-Seq with NuQuant library preparation kit.
The NuQuant workflow for quantification of library molar concentration. Library molarity can be directly measured in less than 6 minutes using a Qubit with the appropriate NuQuant App or a standard fluorescent plate reader
A set of 8 libraries were distributed to 6 different users at multiple institutions. For each library and quantification method, the percent coefficient of variation (% CV) of molar concentration was calculated. NuQuant demonstrated the lowest variation across all users
Sixteen libraries with varying insert sizes and concentrations were quantified with NuQuant. The libraries were then pooled by volume and sequenced. The graph shows the strong correlation between library molarity measured by NuQuant and the number of reads per sample demonstrating the accuracy of NuQuant
Illumina NovaSeq, HiSeq, MiSeq, NextSeq, MiniSeq
Total RNA, poly(A) selected RNA
10 ng - 250 ng
Up to 192 plex with Unique Dual Index Adapters
The Universal RNA-Seq kit provides all necessary buffers, adaptors, primers and enzymes for library preparation. The kit also provides nuclease-free water for purification elution steps. AnyDeplete probes are available separately or in a bundle with the Core Kit. For custom AnyDeplete probes, please contact Tecan NGS Technical Support. Agencourt beads, HL-dsDNase for optional integrated DNase treatment and EvaGreen for optional qPCR must be purchased separately.
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.
The Universal RNA-Seq kit 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:
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.
This system has been designed for use with a broad range of different organisms using organism-specific probes to target specific transcripts for depletion. AnyDeplete probe mixes for human (rRNA), mouse (rRNA plus globin) and Drosophila (rRNA) are available. Please contact Tecan NGS Technical Support for a list of available custom AnyDeplete designs or to design a new custom AnyDeplete probe mix.
The Universal RNA-Seq with NuQuant kit 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 increasing the amount of total RNA input to 100-250 ng. With highly degraded samples, users may experience lower yields and may encounter affected sequencing metrics.
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 technology integrated in the workflow. Custom globin depletion is available for several mammalian species. Contact Tecan NGS Technical Support for more information.
We recommend 10-100 ng high quality RNA, 100-250 ng degraded of 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.
Yes, contaminating genomic DNA can interfere with accurate RNA quantification and may be incorporated into libraries. For this reason we recommend DNase treatment during RNA purification.
For samples that have not undergone a thorough DNase treatment during RNA purification or is suspected of containing contaminating genomic DNA, we recommend starting the protocol at section V.A. First Strand cDNA Synthesis with Integrated DNase Treatment.
For an explanation of DNase requirements see section III.A of the User Guide.
No. The cDNA is generated with random and poly(T) primers, but no SPIA-based amplification is used.
We recommend using the Covaris instrument for cDNA fragmentation, as suggested in the “Materials” section of this user guide. Tecan does not provide the reagents used in the fragmentation steps.
We have evaluated only Covaris fragmented DNA during the development of these systems. Other sonication instruments 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.
Yes, fragmentation can be omitted from the protocol. Note that with high quality input samples, RNA depletion may be less efficient and transcript coverage may be biased towards the 5’ end. Contact Tecan NGS Technical Support for additional information.
The Universal RNA-Seq kit utilizes targeted degradation of an incorporated modified nucleotide to ensure library inserts all carry the same directionality.
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.
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 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:
We recommend using NuQuant to accurately quantify the final libraries for multiplex pooling using a Qubit instrument. The final library pool concentration should be determined using a qPCR-based method before loading onto an Illumina sequencer. Alternatively, use standard NGS library QC methods to quantify your library or refer to the Illumina “Denature and Dilute Libraries Guide” for your specific sequencer.
The single index adaptors add 124 bp to the library and the UDI adaptors add 144 bp to the library.
Universal RNA-Seq libraries are compatible with Illumina sequencing platforms.
Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.
The Universal RNA-Seq is designed for use with the standard Illumina sequencing primers for both single- and paired-end sequencing.
Yes. The libraries produced using this kit can be used for both single- and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The workflow generates libraries with an average insert size that is dependent on the fragmentation conditions used.
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.
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 Universal RNA-Seq kit, the forward read corresponds to the sense strand.
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).
Streamlined solution for total RNA-Seq library preparation for sequencing on Illumina ...
Streamlined solution for mRNA-Seq library preparation for sequencing
For research use only. Not for use in diagnostic procedures.