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Universal Plus™ Total RNA-Seq library preparation kit with NuQuant®

Streamlined solution for total RNA-Seq library preparation for sequencing on Illumina sequencers. This kit features a broad input range, UDI adaptors, rRNA depletion with AnyDeplete® and simple library quantification with NuQuant.
Overview
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Tab 01 / Overview
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Product News: 384 UDI adaptors available now!

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

 

Selection guide

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

Total RNA sequencing (RNA-Seq) is data-rich and enables the collection of the full transcriptome for analysis, unlike conventional technologies such as microarrays that are limited to predefined transcripts. Total 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 Plus Total RNA-Seq with NuQuant library prep kit provides a streamlined solution for whole transcriptome, stranded RNA-Seq from a broad range of inputs including degraded FFPE samples. This kit features a robust chemical fragmentation technology optimized for generating larger insert fragments. Libraries with larger insert sizes reduces the amount of overlapping, redundant sequencing, yielding more unique sequencing data from pair-ended sequencing runs. This RNA-Seq library preparation kit comes 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 Total RNA-Seq library preparation kit include:

  • RNA Sequencing solution for a vast range of sample quality from high-quality RNA to degraded FFPE samples
  • Optimized to generate libraries with larger insert size for sequencing unique and relevant information
  • Identify PCR duplicates by molecular tag
  • Detect index hopping with unique dual indexes (UDI) and increase multiplexing with up to 384 pre-plated adaptors
  • Removal of unwanted transcripts with customizable AnyDeplete
  • Increased accuracy of library concentrations quantification using NuQuant
  • Works across a broad range of RNA inputs (10 – 500 ng)

Tecan kits come standard with all necessary reagents for the entire process from start to finish, including adaptors. Nothing else to buy, streamlining NGS prep to be faster and more cost-effective.

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

Applications

  • Whole transcriptome profiling
  • Gene expression analysis
  • Transcript discovery
  • Splice variant and isoform analysis




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Tab 02 / Highlights
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Optimized protocol to produce libraries with larger insert size so that you are sequencing unique, relevant information.

  • No protocol optimization needed for larger insert size.
  • Obtain better splice junction coverage.
  • Get more data by eliminating redundant, overlapping sequencing of the insert during paired end sequencing runs.

Streamlined workflow for total RNA-Seq

The Universal Plus Total RNA-Seq with NuQuant library preparation kit is a complete solution for total RNA-Seq with a number of key benefits.

  • This kit provides a streamlined workflow with all the reagents for library construction, including UDI adaptors, to go from total RNA to quantified library.
  • Consistent performance and results across a broad input range provides confidence in all of your studies.
  • Eliminate unwanted rRNA reads with AnyDeplete after library preparation.
  • Compatible with both high-quality and degraded FFPE samples.
Show more

Simple streamlined workflow

The Universal Plus Total RNA-Seq with NuQuant kit has a simple streamlined workflow for whole transcriptome RNA-Seq library preparation. The kit provides all the reagents to go from total RNA to quantified libraries ready for Illumina sequencing.

Consistent libraries with as little as 10 ng

Libraries constructed with 10 ng or 500 ng K562 total RNA provides similar metrics minimizing concern about data quality regardless of input amount.

High correlation of sequencing data across the input range

FPKM correlation of the sequencing data from 10 ng and 500 ng inputs showed an R value of 0.94, indicating similar data regardless of the 50-fold difference in input amounts.

Universal Plus Total is compatible with FFPE samples

Libraries were constructed with 100 ng of total RNA isolated from a 7 year old liver FFPE block which had a DV200 of 64%. The sequencing results showed an alignment of over 95% and high % exon but, as expected for FFPE samples, higher % rRNA and intronic reads were also observed. This data demonstrates that Universal Plus Total RNA-Seq with NuQuant is compatible with FFPE samples and provides high quality data.

High quality gene detection

The number of RefSeq genes detected with an FPKM > 1 is similar between the Universal Plus Total RNA-Seq with NuQuant kit, Roche KAPA Stranded RNA-Seq Kit with RiboErase or NEB NEBNext Ultra II Directional RNA Library Prep Kit for Illumina allowing comparisons between data sets. All libraries were generated with 10 ng of K562 total RNA and sequenced on an Illumina platform.

Efficient depletion of rRNA with AnyDeplete

AnyDeplete efficiently depletes human and mouse rRNA to increase dynamic range, reduce sequencing costs, and simplify data analysis.

  • Streamlined workflow to eliminate unwanted transcripts after library construction.
  • Efficient AnyDeplete probe mixes for human and mouse rRNA depletion.
  • Custom AnyDeplete probes available from our expanding list of organisms or design your own.
Show more

AnyDeplete effectively eliminates unwanted ribosomal RNA

Libraries were generated from 100 ng K562 total RNA with and without depletion of human rRNA. In the absence of depletion, rRNA represented approximately 82% of the total sequenced reads. AnyDeplete reduces the percent of rRNA reads to below 1% provide more informative reads from Illumina sequencing.

AnyDeplete can be customized for any organism

Custom depletion allow optimized Illumina sequencing with minimal unwanted reads from any organism. Contact your local sales representative to learn more or order these custom AnyDeplete probe sets.

Unique features designed to save money

NuQuant is a proprietary quantification method providing direct measurement of library molar concentration using a Qubit or standard fluorometers. NGS library quantification with NuQuant is accurate and easy to use.

  • Eliminate the need for time-consuming qPCR and fragment analysis necessary for Illumina sequencing.
  • Accurate measurement of library molar concentration regardless of library size or distribution.
  • Simplify automated quantification and library multiplexing.




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platforma

Compatible Platforma

Illumina NovaSeq, HiSeq, MiSeq, NextSeq, MiniSeq

Starting Material

Starting Material

Total RNA

Input Range

Input Range

10 ng - 500 ng

Multiplexing

Multiplexing

Up to 384-plex with Unique Dual Index adaptors




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Tab 04 / Faq
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FAQ

Getting started

What materials are provided with Universal Plus Total RNA-Seq with NuQuant?

The Universal Plus Total RNA-Seq with NuQuant kit bundle (Part No. 9156, 9157, 9158) provides all necessary buffers, primers and enzymes for fragmentation, cDNA synthesis, library construction, AnyDeplete and NuQuant. SPRI purification beads and EvaGreen are not included.

What is the concentration of the control RNA?

The control RNA (K562 total RNA), included only with the 24 reaction kit, 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?

Universal Plus Total 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.

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

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 Total RNA-Seq with RNA from any organism?

Universal Plus Total RNA-Seq has been designed for use with total RNA inputs from any organism. Custom depletion of rRNA or other transcripts can be designed for any organism. Please contact Tecan NGS Technical Support (techserv-gn@tecan.com) for more information.

Do I need to use high-quality total RNA?

The Universal Plus Total RNA-Seq with NuQuant kit is designed for whole transcriptome RNA-Seq and will work well with high-quality total RNA. The kit has also been shown to be compatible with degraded samples such as FFPE. Contact Tecan NGS Technical Support for more information.

Can contaminating genomic DNA interfere with Universal Plus Total RNA Seq?

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

General workflow

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.

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.

Custom AnyDeplete

Custom depletion designs can be tailored to any transcript, any organism. Please contact Tecan NGS Technical Support at techserv-gn@tecan.com for more information on available existing designs or to develop a new custom design.

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?

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.

Please refer to section IV.N. for guidelines on alternative library quantitative and qualitative assessments.

How many bases do Universal Plus Total RNA-Seq adaptors add to the library?

The adaptors add 144 bp to the library.

Sequencing recommendations

What sequencers are compatible with your libraries?

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

Can Universal Plus Total RNA-Seq libraries be used with paired-end sequencing?

Yes. The libraries produced using this kit 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 workflow generates libraries with an average insert size of approximately 300 bases. Contact Tecan NGS Technical Support for additional information.

Data analysis

What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?

The barcodes provided in the Universal Plus UDI 96-Plex Adaptor Plate (S02480) are a minimum edit distance of 3 from other barcodes in the adaptor plate. This means that a minimum of three edits (replacement, insertion, or deletion) must occur before one barcode becomes a different barcode. The barcodes provided in the Universal Plus UDI-B 96-Plex Adaptor Plate (S02690) are a minimum edit distance of 2 from other barcodes in the adaptor plate. 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 Total RNA-Seq, the forward read corresponds to the sense strand.




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Tab 05 / Literature
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Resources

Type
Title
No
User Guide
Universal Plus Total RNA-Seq with NuQuant
M01523 V3
Product Sheet
400985 V1.1
Product Sheet
Universal Plus Total RNA-Seq
401632 V2
Safety Data Sheet
 




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Tab 06 / Shop
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Order list

 

Product

Part Number

Number of Reactions

Barcodes

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156-24

24

1-24

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156-A01

96

1-96

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156B-A01

96

97-192

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156C-A01

96

193-288

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156D-A01

96

289-384

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157-24

24

1-24

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157-A01

96

1-96

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157B-A01

96

97-192

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157C-A01

96

193-288

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157D-A01

96

289-384

Universal Plus Total RNA-Seq with NuQuant, Custom AnyDeplete

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Product

Part Number

Number of Reactions

Barcodes

Item Price

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156-24

24

1-24

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156-A01

96

1-96

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156B-A01

96

97-192

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156C-A01

96

193-288

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Human AnyDeplete

9156D-A01

96

289-384

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157-24

24

1-24

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157-A01

96

1-96

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157B-A01

96

97-192

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157C-A01

96

193-288

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Mouse AnyDeplete

9157D-A01

96

289-384

Login to see price

Universal Plus Total RNA-Seq with NuQuant, Custom AnyDeplete

--

--

--

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