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Universal Plus™ mRNA-Seq library preparation kit with NuQuant®

Streamlined solution for mRNA-Seq library preparation for sequencing on Illumina sequencers. This kit features a broad input range, UDI, and simple library quantification with NuQuant.




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Tab 01 / Overview
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Product News: 384 UDI adaptors available now!

The Universal Plus mRNA-Seq with NuQuant is now available with up to 384 UDI (Unique Dual Index) adaptors for maximum multiplexing flexibility to optimize your sequencing runs.

From RNA samples to quantified libraries for mRNA-Seq studies in a single workflow

Standard whole transcriptome RNA-Seq captures both informative and non-informative data. mRNA-Seq is a more targeted approach for studying the transcriptional coding regions and capturing highly informative gene expression data that can be used in multiple applications.

mRNA-Seq is used to examine transcriptional expression in a wide variety of research areas, including disease conditions. mRNA-Seq is a selective RNA-Seq procedure that enriches for all poly(A) transcripts within the transcriptome. Targeting mRNA maximizes sequencing coverage since resources are dedicated to the sequencing of coding regions. This enables the detection of rare variants and low expressed mRNA transcripts.

Universal Plus mRNA-Seq kit with NuQuant® library quantification is a novel and streamlined library preparation solution for mRNA sequencing on Illumina sequencers. This kit is designed to provide poly(A) selection efficiency and uniform coverage for a broad range of RNA inputs. 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. The NuQuant method eliminates the need for time-consuming or inaccurate library quantification methods like qPCR, fluorometry (e.g. Qubit®) or microfluidic electrophoresis (e.g. Bioanalyzer®) allowing RNA-Seq libraries to be constructed and quantified in a single day.

Benefits of Universal Plus mRNA-Seq library preparation kit include:

  • Identify PCR duplicates by molecular tag
  • Detect index hopping with unique dual indexes (UDI) and increase multiplexing with up to 384 pre-plated adaptors
  • Fewer bead purification steps for an efficient and streamlined library preparation workflow for any sample type
  • Broad dynamic input range from 1 μg to as low as 10 ng, enables Illumina sequencing of previously inaccessible samples
  • Eliminate adaptor dimers without adaptor titration regardless of sample input using the DimerFree® technology
  • Accurately quantify library concentrations (in nM) with a simple fluorescent measurement using NuQuant library quantification method at no additional cost
  • Remove unwanted reads from sequencing data using AnyDeplete® mediated transcript depletion after library preparation (Learn more in our Application Note)

NuQuant app for Qubit® can be downloaded here on Github.

Applications

  • mRNA sequencing
  • Gene expression analysis
  • Transcript discovery
  • Splice variant and isoform analysis
  • Fusion detection




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Tab 02 / Highlights
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NuQuant Library Quantification Method: Accurately quantify molar concentrations of NGS libraries

NuQuant Library Quantification provides a simple library quantification that saves time and money. During library preparation, 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. NGS library quantification with NuQuant is accurate, easy to use and is integrated with the Universal mRNA-Seq with NuQuant library preparation kit.

  • Accurately measure molar library concentration without the need for library size analysis (e.g. Bioanalyzer) unlike Qubit mass measurement or qPCR
  • Quantify only sequenceable molecules unlike Qubit and Bioanalyzer
  • Eliminate the need of time consuming qPCR for library quantification
  • Compatible with standard fluorometers such as the Qubit or standard plate readers
Show more

NuQuant is more accurate than traditional methods

NuQuant has the lowest variability during library quantitation by multiple users. A set of 8 libraries was distributed to 6 different users at multiple institutions. Users were asked to use NuQuant and their own preferred method of library quantitation (number denoted by n). For each library, and each quantification method, the percent coefficient of variation (%CV) of molar concentration was calculated. NuQuant produced the lowest variation, followed by qPCR. Bioanalyzer gave the highest variation.

NuQuant determined library molarity

Strong correlation between NuQuant molarity and sequencing read numbers. Two users prepared 8 libraries each from 10 ng of Covaris fragmented genomic DNA and amplified the libraries with either 10 or 13 cycles to produce both lower and higher library concentrations. The 16 purified libraries were quantified by NuQuant and equal volumes of all libraries were pooled and sequenced on one lane of an Illumina sequencer.  The number of reads from each library correspond to the library molarity determined by NuQuant demonstrating the accuracy of quantification.

NGS library quantification workflow with NuQuant method

NuQuant simplifies library quantification. Using any standard fluorometer, NuQuant generates library molarity values that can be used for pooling NGS libraries before sequencing.

Library quantification workflow with NuQuant compared to qPCR, Qubit and Bioanalyzer.

NuQuant saves time and money by eliminating the need for qPCR and bioanalyzer steps and reagents.

Reproducible mRNA-Seq library preparation and sequencing results

  • Simple one-day workflow from total RNA to quantified RNA-Seq libraries for Illumina sequencers
  • Consistent results across a wide range of inputs from 10 ng to 1μg of total RNA
  • Uniform coverage across the entire transcript
Show more

Consistent libraries even with as little as 10 ng input RNA

Universal Plus mRNA-Seq library preparation kit with NuQuant was used to prepare Illumina libraries starting with 10 ng, 100 ng and 1 μg of K562 total RNA. Consistent library metrics regardless of input was observed. This allows for RNA sequencing of low abundant samples that were previously inaccessible.

Gene detection is similar regardless of input

Universal Plus mRNA-Seq library preparation kit with NuQuant provides consistent results regardless of input. Similar detection of RefSeq genes even at lower inputs, demonstrating robust performance and high quality data regardless of input.

Even gene coverage enables better splice junction and isoform analysis

Universal Plus mRNA-Seq library prep kit with NuQuant provides even coverage across the entire transcript enabling detection of splice variants and isoforms during data analysis.

Remove unwanted reads with AnyDeplete

  • Streamlined workflow to eliminate abundant unwanted reads (i.e. globin, mitochondrial transcripts, rubisco, etc.)
  • Simple customization of AnyDeplete probe design to optimize your experiment
  • Simplify data analysis and reduce sequencing costs

Universal Plus mRNA-Seq library prep kit was used to construct Illumina libraries from 500 ng of total RNA from fetal cord blood. In the absence of AnyDeplete-mediated globin transcript depletion, globin represented ~77% of the sequencing reads.

Addition of adult globin depletion reduced the percent of globin down to ~43%, increasing the number of informative reads. The AnyDeplete probe mix was customized to target fetal globin which reduced the goblin to ~10%. Eliminating unwanted globin reads resulted in the detection of over a 1000 more RefSeq genes providing more complete data.




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

Illumina HiSeq, MiSeq, NextSeq, MiniSeq, NovaSeq

Starting Material

Starting Material

Total RNA

Input Amounts

Input Amounts

10 ng - 1 μg

Multiplexing

Multiplexing

Up to 384-plex with Unique Dual Index adaptors




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Tab 04 / Faq
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FAQ -> Universal Plus RNA-Seq with NuQuant

What materials are provided with Universal Plus mRNA-Seq?

The Universal Plus mRNA-Seq core kit provides all necessary buffers, primers and enzymes for poly(A) selection and library construction. SPRI purifcation beads and EvaGreen are not included. Reagents for AnyDeplete are available separately.

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?

Universal Plus mRNA-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.

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

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 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.

Can I use Universal Plus mRNA-Seq with RNA from any organism?

Universal Plus mRNA-Seq has been designed for use with total RNA inputs containing polyadenylated transcripts. Universal Plus mRNA-Seq is compatible with polyadenylated RNA from any organism.

Do I need to use high-quality total RNA?

Yes, high-quality RNA is required for efficient poly(A) selection.

Can contaminating genomic DNA interfere with Universal Plus mRNA Seq?

Contaminating genomic DNA can be incorporated into libraries and inclusion of a DNase treatment during RNA isolation is recommended.

Does this system contain a SPIA®-based amplification?

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. Chemical fragmentation is incorporated into the workflow after poly(A) RNA selection.

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.

Where can I safely stop in the protocol?

Samples can be placed in short-term storage at –20 °C after second strand synthesis, end repair, strand selection or after any of the bead purification steps.

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.

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 Universal Plus mRNA-Seq adaptors add to the library?

Single index adaptors add 122 bp to the library. Dual index adaptors add 144 bp to the library.

What sequencers are compatible with your libraries?

Universal Plus mRNA-Seq libraries are compatible with Illumina sequencing platforms.

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.

What kind of sequencing primers can I use with your libraries?

Universal Plus mRNA-Seq libraries are designed for use with the standard Illumina sequencing primers for both single- and paired-end sequencing.

Can Universal Plus mRNA-Seq libraries be used with 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. Contact Tecan NGS Technical Support for additional information.

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.

Can I use standard alignment algorithms to analyze strand-specific sequencing data?

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 Universal Plus mRNA-Seq, the forward read corresponds to the sense strand.




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Tab 05 / Literature
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Literature

Type
Title
No
Application Note
401103 V1
Application Note
401099 V1
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For research use only. Not for use in diagnostic procedures.