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Universal RNA-Seq Library Preparation Kit with NuQuant®

Robust solution for stranded whole transcriptome RNA-seq library prep for any sample type and RNA quality
Overview
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Tab 01 / Overview
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Product News

We have recently introduced Universal Plus Total RNA-Seq with NuQuant for whole transcriptome RNA-Seq without time-consuming mechanical fragmentation.

 

Selection guide

Whole transcriptome RNA sequencing library prep and quantification in a single workflow

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:

  • Compatible with high-quality RNA and degraded FFPE samples
  • Works across a broad range of RNA inputs (10 - 250 ng)
  • Increased accuracy of library concentration quantification using NuQuant
  • Remove unwanted transcripts from any organism with AnyDeplete® technology
  • Detect index hopping with up to 192 Unique Dual Index (UDI) adaptors

Applications

  • Whole transcriptome profiling
  • Gene expression
  • RNA sequencing
  • Transcript discovery
  • Splice variant detection and discovery

 




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Tab 02 / Highlights
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Simple, streamlined workflow for any sample type

The Universal RNA-Seq with NuQuant library preparation kit is designed to provide consistent data and performance from any organism and sample type.

The kit provides all the reagents for library construction and library quantification.

RNA-Seq Library Prep Nuquant

Ovation Whole Blood Signal Correlation

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.

Efficient depletion of unwanted transcripts with AnyDeplete

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.

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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.

  • Streamlined workflow to eliminate unwanted transcripts after library construction
  • Validated AnyDeplete probe mixes for human, mouse and Drosophila rRNA depletion
  • Custom AnyDeplete probes available from our expanding list of organisms or design your own
  • Mix AnyDeplete probes together to target multiple transcripts and species

Efficiently eliminate unwanted transcripts

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

Validated AnyDeplete probes for mouse, drosophila and custom rRNA depletion

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 AnyDeplete probes

Available custom AnyDeplete probes for Universal RNA-Seq with NuQuant library prep kit

Simple library quantification with NuQuant

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.

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NGS library quantification with NuQuant is accurate, easy to use and is integrated with the Universal RNA-Seq with NuQuant library preparation kit.

  • Eliminate the need for time consuming qPCR and fragment analysis for library quantification
  • Accurate measurement of library molar concentration regardless of library distribution
  • Quantify only sequenceable molecules using a Qubit or any standard plate reader

Library quantification workflow with NuQuant

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

NuQuant has the lowest variability for library quantification

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

Strong correlation between NuQuant molarity and read numbers

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




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

Illumina NovaSeq, HiSeq, MiSeq, NextSeq, MiniSeq

Starting Material

Starting Material

Total RNA, poly(A) selected RNA

Input Range

Input Range

10 ng - 250 ng

Multiplexing

Multiplexing

Up to 192 plex with Unique Dual Index Adapters




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Tab 04 / Faq
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FAQ

Getting started

What materials are provided with the Universal RNA-Seq with NuQuant?

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.

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?

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.

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.

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

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.

Do I need to use high-quality total RNA?

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.

Do I need to perform an rRNA depletion, poly(A) enrichment step, or globin depletion before processing total RNA samples with the Universal RNA-Seq with NuQuant kit?

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.

How much total RNA do I need for library generation?

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.

Can contaminating genomic DNA interfere with the Universal RNA-Seq with NuQuant performance?

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.

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.

Does Tecan provide reagents for performing the fragmentation step of the protocol?

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.

I don’t have access to a Covaris instrument. Can I use alternative fragmentation methods?

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.

Can I skip mechanical fragmentation before cDNA concentration?

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.

How does your protocol enable strand retention?

The Universal RNA-Seq kit utilizes targeted degradation of an incorporated modified nucleotide to ensure library inserts all carry the same directionality.

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, after cDNA fragmentation 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. 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.

How many bases do the Universal RNA-Seq with NuQuant adaptors add to the library?

The single index adaptors add 124 bp to the library and the UDI adaptors add 144 bp to the library.

Sequencing recommendations

What sequencers are compatible with your libraries?

Universal 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?

The Universal RNA-Seq is designed for use with the standard Illumina sequencing primers for both single- and paired-end sequencing.

Can the Universal RNA-Seq 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. The workflow generates libraries with an average insert size that is dependent on the fragmentation conditions used.

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.

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 the Universal RNA-Seq kit, the forward read corresponds to the sense strand.

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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Literature

For research use only. Not for use in diagnostic procedures.
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