Expression Profiling Kits

Reagents & Kits

Expression Profiling/QuantSeq Expression Profiling Library Prep Kits - Lexogen

QuantSeq kits enable cost-efficient sequencing by counting. These kits are an exceptional alternative to standard RNA-Seq and microarrays. Just one fragment per transcript is produced and therefore there is no need for length normalization. This makes data analysis very simple and accurate.

The kits of Lexogen are available in 2 versions and for different platforms. 

The QuantSeq FWD kit generates reads towards the poly-A tail, directly reflecting the mRNA sequence. This version is available for Illumina and IonTorrent platforms.

The QuantSeq REV kit enables exact pinpointing the 3’ end in Read 1, as the first nucleotide of the read corresponds to the very last nucleotide of the mRNA sequence. This requires a Custom

Sequencing Primer (CSP version 2, included in the kit). This version is only available for Illumina platforms.

12nt UDI Indexing kits  are available to increase the number of libraries that can be pooled on a single lane of an Illumina Flow cell.
•    QuantSeq 3’ mRNA-Seq Library Prep Kit FWD (Illumina/IonTorrent)
•    QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina
•    QuantSeq-Flex Targeted RNA-Seq Library Prep Kit v2 for Illumina
•    QuantSeq Modules & Add-ons 

 

QuantSeq 3’ mRNA-seq Library Prep Kit FWD (Illumina/IonTorrent)


The QuantSeq FWD kit is designed to generate Illumina or IonTorrent compatible libraries of sequences close to the 3’ end of poly-A RNA
o    Genome-wide analysis of gene expression
o    Cost-saving and streamlined globin mRNA depletion during library prep
o    Cost-efficient alternative to microarrays and standard RNA-seq
o    Down to 100pg total RNA input (5ng for IonTorrent version)
o    Applicable for low quality and FFPE samples
o    Sequencing ready libraries in 4.5hours
o    Free Data analysis on the Bluebee® Genomics Analysis Platform

 

Analysis of Low Input and Low Quality Samples

QuantSeq kits are compatible with low input samples, down to 100pg and are suited to work with low quality input material, including FFPE samples.

Figure 2: Correlation of gene counts of FFPE and cryo samples

figure 1: Venn diagrams of genes detected by QuantSeq at a uniform read depth of 2.5M reads in FFPE and Cryo samples with 1, 5 and 10 reads/gene thresholds

High Strand Specificity

QuantSeq maintains exceptional high strand-specificity (>99.9%) and allows to map reads to their corresponding strand on the genome, enabling the discovery and quantification of antisense transcripts and overlapping genes.

Mapping of Transcript End Sites (TES)
By using longer reads, QuantSeq FWD allows to exactly pinpoint the 3’ end of poly-A RNA and therefore obtain accurate information about the 3’ UTR.

figure 3: QuantSeq Read coverage vs normalized transcript length of NGS libraries derived from FFPE-RNA (blue) and cryo-RNA (red)

Unique Molecular Identifiers (UMI)
The separately available UMI Second Strand Synthesis Module for QuantSeq FWD allows unique tagging of individual transcripts with 6nt long UMI’s, located between the partial P5 adapter and the random priming sequence. This enables identifying PCR duplicates and eliminating amplification bias.

Direct Counting for Gene Expression Quantification
QuantSeq kits produce only 1 fragment per transcript. Therefore, no length normalization is required, allowing more accurate determination of gene expression values. This makes QuantSeq the best alternative to microarrays and conventional RNA-seq in Gene Expression studies.

Rapid Turnaround
In only 4.5 hours, including less then 2 hours hands-on time, QuantSeq’s straightforward workflow generates sequencing-ready NGS libraries

Cost Saving Multiplexing
The length restriction in QuantSeq libraries allows a high level of multiplexing by saving sequencing space and enables up to 9.216 libraries being sequenced in a single Illumina lane.

Automation Friendly
The QuantSeq kit is available as an automated version (autoQuantSeq 3’ mRNA-seq Library Prep kit for Illumina) as well to accommodate automated library prep. The protocol has been adapted for automation on several platforms (including Sciclone NGS, Hamilton Microlab STAR, Bravo and Biomek platforms). 

Simple Bioinformatics Analysis
The QuantSeq data analysis pipeline has been integrated on the Bluebee Genomics Analysis Platform and can be used by any user, even without bioinformatics background. The access codes are included in QuantSeq kits and additional codes can be purchased separately.Ordering information

Ordering information

Contact us for pricing and additional information 

Cat. n°

Description

Reaction size

015.24

QuantSeq 3' mRNA-seq Library Prep Kit FWD for Illumina

24 preps

015.96

QuantSeq 3' mRNA-seq Library Prep Kit FWD for Illumina

96 preps

015.2x96

QuantSeq 3' mRNA-seq Library Prep Kit FWD for Illumina

2x 96 preps

015.384

QuantSeq 3' mRNA-seq Library Prep Kit FWD for Illumina HT  including i5 DI Add-on Kit (5001-5004)

384 preps

012.24A

QuantSeq 3' mRNA-seq Library Prep Kit for IonTorrent, Barcode Set A (01-24)

24 preps

012.24B

QuantSeq 3' mRNA-seq Library Prep Kit for IonTorrent, Barcode Set B (25-48)

24 preps

Modules and Add-ons for QuantSeq Kits

click here

090.24

QuantSeq Data Analysis (FWD/FWD-UMI) on Bluebee Genomics Platform

0-1.5 Gb

093.03

QuantSeq Data Analysis (FWD/FWD-UMI) on Bluebee Genomics Platform, 1 run

0-3 Gb

093.06

QuantSeq Data Analysis (FWD/FWD-UMI) on Bluebee Genomics Platform, 1 run

0-6 Gb

 

QuantSeq 3’ mRNA-seq Library Prep Kit REV for Illumina


The QuantSeq REV kit is designed to generate libraries at the 3’ end of poly-A RNA. This makes it possible to exactly pinpoint the 3’ end in Read 1 and enables the study of the 3’ UTR and alternative polyadenylation.
•    3’ UTR analysis and study of alternative polyadenylation
•    Down to 10ng total RNA input, including FFPE samples
•    Sequencing ready libraries in 4.5 hours with only 2 hours hands-on time
•    Cost-efficient sequencing of up to 9.216 samples/lane
•    Automation friendly
•    Free Data-analysis via the Bluebee® genomics analysis platform

Depletion of globin mRNAs during QuantSeq Library Prep
Combined with the Globin Block Module, it is possible to generate globin-depleted, sequencing ready 3’ mRNA libraries from as little as 50ng of total RNA frow whole blood. The Globin Block module can be integrated seamlessly in the protocol without additional protocol steps. Using this module, reads mapping to globin mRNA can be reduced to as low as 5%, dramatically increasing gene detection.

figure 5: Percentage of sequencing reads mapping to globin mRNAs rom human (Hs) and pig (Ss) blood QuantSeq libraries *

4/12figure 4: Increased gene detection in human blood QuantSeq libraries using Globin Block. CPM = Counts per million*

High Strand-specificity
While maintaining exceptional strand-specificity of 99,9%, QuantSeq allows to map reads to their corresponding strand on the genome. This enables the discovery and quantification of antisense transcripts and overlapping genes.


Direct Counting for Gene Expression Quantification
Since only 1 fragment per transcript is produced, QuantSeq allows more accurate determination of gene expression values. Therefore, QuantSeq kits are the best alternative to microarrays and conventional RNA-seq in Gene Expression and eQTL studies.

Cost Saving Multiplexing
Since the length restriction in QuantSeq saves sequencing space, the libraries are intended for a high degree of multiplexing. With up to 96 i7 indices included in the kit and the additionally available 96 i5 indices, up to 9.126 samples can be multiplexed and loaded on a single lane, resulting in a substantial saving on sequencing costs.

Simple Bioinformatics analysis
The QuantSeq data analysis pipeline has been integrated on the Bluebee Genomics Analysis Platform and can be used by any user, even without bioinformatics background. The access codes are included in QuantSeq kits and additional codes can be purchased separately.

Automation Friendly
The QuantSeq kit is available as an automated version (autoQuantSeq 3’ mRNA-seq Library Prep kit for Illumina) as well to accommodate automated library prep. The protocol has been adapted for automation on several platforms (including Sciclone NGS, Hamilton Microlab STAR, Bravo and Biomek platforms). 

Ordering information

Contact us for pricing and additional information

Cat. n°

Description

Reaction size

016.24

QuantSeq 3' mRNA-seq Library Prep Kit REV for Illumina with Custom Sequencing Primer

24 preps

016.96

QuantSeq 3' mRNA-seq Library Prep Kit REV for Illumina with Custom Sequencing Primer

96 preps

016.2x96

QuantSeq 3' mRNA-seq Library Prep Kit REV for Illumina with Custom Sequencing Primer

2x 96 preps

Modules and Add-ons for QuantSeq Kits

click here

091.24

QuantSeq Data Analysis (REV) on Bluebee Genomics Platform

0-1.5 Gb

094.03

QuantSeq Data Analysis (REV) on Bluebee Genomics Platform, 1 run

0-3 Gb

094.06

QuantSeq Data Analysis (REV) on Bluebee Genomics Platform, 1 run

0-6 Gb

 

QuantSeq-Flex Targeted RNA-seq Library Prep Kit V2 for Illumina

The QuantSeq-Flex kit is a library preparation kit, designed to make Illumina compatible liraries from any RNA sample, using custom primers.  Whether it is gene expression analysis, targeted sequencing, adapter-ligation based RNA-seq or any other experiment where a certain region needs to be sequenced, QuantSeq-Flex can be used together with user-supplied primers.

The following types of libraries can be generated:
1.    OligodT primed in reverse transcription, random primed in second strand synthesis (QuantSeq 3’ mRNA-seq)
2.    OligodT primed in reverse transcription, target-specifically primed in second strand synthesis (targeted 3’ mRNA-seq)
3.    Target-specifically primed in reverse transcription, random primed in second strand synthesis (Targeted RNA-seq, allowing identification of novel fusions)
4.    Target-specifically primed in reverse transcription, target-specifically primed in second strand synthesis (targeted RNA-seq; detecting known targets only

•    Flexible kit for targeted sequencing and molecular barcoding
•    Identification of known and unknown Fusion Transcripts
•    Use of custom-designed gene-specific primers for first and/or second strand synthesis
•    Cost-efficient and flexible alternative to pre-designed sequencing panels
•    Multiplexing of up to 9.126 samples per lane

An RNA-Seq Experiment Tailored to your needs
Quant-Seq Modular Kits give the needed flexibility to enable implementation of RNA-seq as part of the experimental pipeline. One Fragment per transcript produced and the use of custom primers makes this kit a perfect solution for cost-saving high-throughput gene expression analysis. Quant-Seq Flex is suitable for targeted sequencing of gene panels, enrichment, detection of fusion genes and various other types of transcripts.

Rapid Turnaround
The straightforward protocol allows generating sequencing-ready libraries in only 4.5 hours, including a hands-on time of less than 2 hours.

Cost Saving Multiplexing
QuantSeq Libraries are intended for a high degree of multiplexing. When combining the 96 i7 indices, included in the kit, with the separately available 96 i5 indices, up to 9.216 samples can be combined on a single lane of an Illumina Flow Cell. This allows substantial sequencing cost saving, as the length restriction in QuantSeq saves sequencing space.
 

Ordering information:
Contact us for pricing and additional information

Cat. n°

Description

Reaction size

033.24

QuantSeq-Flex targeted RNA-Seq Library Prep Kit v2 with first strand synthesis Module for Illumina

24 preps

033.96

QuantSeq-Flex targeted RNA-Seq Library Prep Kit v2 with first strand synthesis Module for Illumina

96 preps

034.24

QuantSeq-Flex targeted RNA-Seq Library Prep Kit v2 with second strand synthesis Module for Illumina

24 preps

034.96

QuantSeq-Flex targeted RNA-Seq Library Prep Kit v2 with second strand synthesis Module for Illumina

96 preps

035.24

QuantSeq-Flex targeted RNA-Seq Library Prep Kit v2 with first and second strand synthesis Module for Illumina

24 preps

035.96

QuantSeq-Flex targeted RNA-Seq Library Prep Kit v2 with first and second strand synthesis Module for Illumina

96 preps

026.96

QuantSeq-Flex First Strand Synthesis Module for Illumina

96 preps

028.96

QuantSeq-Flex Second Strand Synthesis Module v2 for Illumina

96 preps

047.4x24

Lexogen i5 6nt Dual Indexing Add-on kit (5001-5004)

4x24 rxn

047.4x96

Lexogen i5 6nt Dual Indexing Add-on kit (5001-5004)

4x96 rxn

047.96

Lexogen i5 6nt Unique Dual Indexing Add-on kit (5001-5096)

96 rxn

 

QuantSeq Modules & Add-ons


Globin Block Modules for QuantSeq (Click to go down)
Dual Indexing (i5 indexing) Modules for QuantSeq (Click to go down)
12nt Unique Dual Index System (UDI) for RNA-seq (Click to go down)
 i7 6nt Index Set (7001-7096) (Click to go down)
UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina) (Click to go down)
PCR Add-on Kit for Illumina (Click to go down)
PCR Add-on Kit for IonTorrent (Click to go down)
Purification Module with Magnetic Beads (Click to go down)

 

•    Globin Block Modules for QuantSeq


The Globin Block (RS-Globin Block) Modules for QuantSeq prevent the generation of library fragments from globin mRNA’s by blocking their extension during second strand synthesis. This module is intended for the preparation of libraries from blood RNA samples. The module is compatible only with QuantSeq 3’ mRNA-seq library prep kits for Illumina FWD (Cat. n° 015.xx) and REV (Cat. n° 016.xx)

Each module provides a modified RNA removal solution (RS-Globin block), containing species-specific globin blocking oligos. The RS-Globin block solution replaces the RNA removal solution from the standard QuantSeq 3’ mRNA-seq library prep kits. Separate modules are available for human (Homo sapiens) and pig (Sus scrofa)

The RS-Globin Block Homo sapiens module (RS-GBHs) is designed for human blood and contains blocking oligo’s against the human alpha en beta hemoglobin chain mRNA’s HBA1, HBA2 and HBB

The RS-Globin Block Sus scrofa module (RS-GBSs) is designed for pig blood and contains blocking oligo’s against the pig alpha and beta hemoglobin chain mRNA’s HBA and HBB.

 

Ordering information:

Cat. n°

Description

Reaction size

070.96

RS-Globin Block module, Homo sapiens

96 preps

033.96

RS-Globin Block module, Sus scrofa

96 preps


Contact us for pricing and additional information 
 

 

•    Lexogen Dual Indexing (i5 indexing) Modules for QuantSeq

The Lexogen i5 6nt Dual Indexing Add-on Kit contains perfectly balanced i5 indices and is available in sets of 4 (5001-5004) and 96 (5001-5096) indices

o    96 i5 unique barcode combinations for fully-flexible Dual Indexing
o    6bp indices with perfect, uniform nucleotide and color balance
o    Hamming distance of 3 enables both error detection and error correction
o    Detect and quantify index hopping
o    Avoid cross-contamination and minimize index mis-assignment
o    Online index balance checker tool  to assist with the choice of the optimal index combination for your experiment.

I5 indices can be introduced at the PCR-step of QuantSeq 3’ mRNA-seq kits. Each of the i5 indices can be combined with any of the 96 i7 indices, included in the QuantSeq kits, enabling up to 9.216 different barcoding options.

Ordering information:

Cat. n°

Description

Reaction size

047.4x24

Lexogen i5 6nt Dual Indexing Add-on kit (5001-5004)

4x24 rxn

047.4x96

Lexogen i5 6nt Dual Indexing Add-on kit (5001-5004)

4x96 rxn

047.96

Lexogen i5 6nt Unique Dual Indexing Add-on kit (5001-5096)

96 rxn

Contact us for pricing and additional information 
 

•    Lexogen 12nt Unique Dual Index System (UDI) for RNA-seq


The 12nt UDI sets are an add-on, introduced at the PCR step of Library Preparation, enabling up to 384 unique combinations for higher multiplexing of sequencing libraries on Illumina Flow cells. The Indices are designed to maximize inter-index distance for different sample numbers and index read-out lengths. Read mis-assignment due to index hopping is avoided and index sequence errors can be corrected with the highest accuracy. This system provides the optimized indexing solution for current and future barcoding requirements.

Effects of Index hopping and Index Sequence Errors

Read mis-assignment caused by Index Hopping can be avoided by using Unique Dual Indexing (UDI). Reads with hopped indices are irreversibly discarded (C). Reads with random Index Sequence Errors resulting in an index not present in the pool are classified undetermined. Accurate error correction can rescue most of these reads making them available for downstream data analysis (B). The percentage values were derived from an RNA-Seq experiment pooling 96 libraries with Lexogen’s 12 nt UDIs and full 12 nucleotide index read-out on an Illumina NextSeq500.

Scalable Index Read-out Length

The design enables scalable read-out lengths of 12, 10 and 8 nucleotides. In ths way, the UDIs support all kinds of requirements for multiplexing, which depend on experiment type, sequencing equipment, desired read depth, and/or the number of pooled libraries. For small sample sizes, short indices (8 or 10nt) are sufficient to ensure high accuracy and reliable error correction. For more than 96 samples however, 8nt index read-out does not allow reliable error correction anymore, and 10 or 12nt read-outs are required.

While needing slightly more sequencing cycles, 12nt index sequences provide the ability to correct up to 3 Index Sequence Errors. Adjustable index read-out length allows tuning your index needs to the experiment design, without the need to purchase separate indexing sets.

Subset

12nt

10nt

8nt

D

ec

D

ec

D

ec

384

4

1

3

1

2

1*

 

L96

5

2

4

1

3

1

 

L24

6

2

5

2

4

1

 

L4

7

3

6

2

5

2

table 1: Inter-index distance (D) and number of errors that can be corrected (ec) are compared for subsets of 384, 96, 24, and 4 libraries and the three possible read-out lengths. For smaller subsets (up to 96 samples) a read-out of 8 or 10 nt allows correction of one error and thus recovery of additional reads. Larger subsets require a read-out of 10 or 12 nt to benefit from the error correction. The ec values represent the number of all errors (including substitutions, insertions, and deletions) that can be confidently corrected, except for *. In this case error correction can only address substitutions.

Nested Index Set Design for Highest Accuracy

 The 12nt UDIs have been designed in a nested approach to provide optimal index subsets for varying multiplex needs. Small subsets benefit from the nested Index system by having the largest inter-index distance and highest error correction capacity, while larger subsets provide for higher multiplexing needs.

All subsets are nucleotide-balanced at each index position for optimized cluster identification in the NGS run. Using a proprietary algorithm, Lexogen has designed more then 9.216 UDIs (24x384 subsets) with the capacity of correcting at least 1 error. Such sets with more than 384 UDIs are available upon request and enable extreme levels of multiplexing, while still providing excellent error correction.

figure 6: Illustration of inter-index distance (D) and number of possible error corrections (ec) in a nested index set with 12 nt read-out. An optimized set of 384 indices contains a subset of 96 indices with larger distances and enhanced error correction. Within these 96 indices, a subset of 24 indices is optimized even further, while a 4 index subset features the highest possible inter-index distance and error correction capacity.

Ordering Information:
Coming Soon 

Contact us for pricing and additional information 

 

•    Lexogen i7 6nt Index Set (7001-7096)


Includes a 96-well plate with 96 i7 indices (7000-7096) that can be used with any QuantSeq Kit


Ordering information:

Cat. n°

Description

Reaction size

044.96

Lexogen i7 6nt Index set (7001-7096) 1rxn/index

96 rxn

Contact us for pricing and additional information 
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•    UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina)


 The UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina) contains the UMI Second Strand Synthesis Mix (USS). This mix simply replaces the Second Strand Synthesis Mix 1 (SS1) from the standard QuantSeq FWD kit.

o    Unique Molecular Identifiers (UMI) for detection and elimination of PCR duplicates
o    Streamlined during QuantSeq FWD Library Preps
o    Free data analysis pipeline on the Bluebee® Genomics Analysis platform

Use this module to identify PCR duplicates and eliminate amplification bias.

Ordering information:

Cat. n°

Description

Reaction size

081.96

UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina)

96 rxn


Contact us for pricing and additional information 
 

 

•    PCR Add-on Kit for Illumina

The PCR Add-on kit contains a PCR mix including an Illumina P5 specific primer, a thermostable polymerase and Illumina P7 primer without a barcode for 96rxn. By adding SYBR Green I to the PCR reaction, a qPCR assay to determine the optimal number of cycles for the endpoint PCR of your QuantSeq libraries can be set up.

The qPCR assay is recommended to determine the exact number of cycles for the endpoint PCR in order to prevent any under- or overcycling of your library. Undercycling may result in too little library, while overcycling can lead to significant distortion in gene expression values.

The kit furthermore contains a reamplification primer that can be used to reamplify already barcoded libraries if they were undercycled to get enough material for sequencing.

Ordering information:

Cat. n°

Description

Reaction size

020.96

PCR Add-on Kit for Illumina

96 rxn


Contact us for pricing and additional information 
 

 

•    PCR Add-on Kit for IonTorrent

 

The PCR Add-on kit contains a PCR Mix including IonTorrent specific primers and a thermostable polymerase. With the PCR Add-on kit more endpoint PCR’s can be run for your sample to determine the exact number of cycles needed to prevent any under- or overcycling of your library. Undercycling may result in too little library while overcycling can lead to significant distortions in gene expression values. If your sample is undercycled, the kit can furthermore be used to add more cycles and reamplify your library.

Ordering information:

Cat. n°

Description

Reaction size

021.96

PCR Add-on Kit for IonTorrent

96 rxn


Contact us for pricing and additional information 

•    Purification Module with Magnetic Beads


The Purification Module contains all the necessary reagents to carry out additional purifications eg. after PCR or to concentrate libraries if needed.

Ordering information:

Cat. n°

Description

Reaction size

022.96

Purification Module with Magnetic beads

200 rxn

Contact us for pricing and additional information 
 

LEARN MORE ABOUT THE EXPRESSION PROFILING/QUANTSEQ EXPRESSION PROFILING LIBRARY PREP KITS

Request a quote or ask your question about our Expression Profiling/QuantSeq Expression Profiling Library Prep Kits via the contact options below. We will try to answer your question within one working day.

CONTACTFORM

Direct contact

+31 30 6880771
info@isogen-lifescience.com

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