mRNA/whole transcriptome sequencing

mRNA/whole transcriptome sequencing

The Lexogen SENSE mRNA/total RNA Library Prep kits have been well established over the last years and have proven to deliver high quality RNA-seq libraries, generating best-in-class results. SENSE kits come in 2 separate versions, so one still has to decide to acquire a kit for mRNA sequencing or a kit for total RNA sequencing.

Lexogen has now launched the CORALL total RNA-seq Library Prep kit. This kit shows improved performance over the SENSE kits, including reduced PCR cycle numbers for library amplification and improved coverage uniformity.

But, in addition, CORALL RNA-seq Library Prep kits succeed in consolidating your RNA-seq workflows, as the CORALL kits can be used for mRNA-seq as well as for total RNA-seq, simply by adding a specific module to treat your input material.

•    CORALL Total RNA-Seq Library Prep Kit
•    SENSE mRNA-Seq Library Prep Kit v2
•    SENSE Total RNA-Seq Library Prep Kit
•    Modules and Add-ons 

CORALL Total RNA-seq Library Prep Kit

CORALL is the all-in-one library prep kit for whole transcriptome sequencing, featuring seamless integration of Unique Molecular Identifiers (UMI), excellent whole transcriptome coverage and exceptional protocol-inherent strand specificity. The fragmentation-free protocol uses Lexogen’s proprietary Strand Displacement Stop and Ligation technologies to deliver complete transcript representation, including start and end sides.

When combined with the Poly-A RNA selection kit, CORALL is the first choice for mRNA-seq
o    Complete coverage of transcripts from start to end
o    Ready-to-sequence libraries within 4.5 hours
o    UMI’s seamlessly included
o    Excellent protocol-inherent strandedness (99%)
o    1ng to 1µg of total RNA input
o    Easy all-in-one protocol
o    Automation Friendly

The kit can be used for the following applications:
o    Gene Expression Profiling
o    Isoform discovery and quantification
o    Alternative splicing studies
o    Transcript (re)annotation
o    SLAMseq metabolic RNA sequencing

Flexible Input
CORALL Library prep supports an input of 1ng to 1µg of total RNA input. However, depending on the sequencing application, a pre-treatment of the input RNA is recommended.

For mRNA-seq, a pre-treatment with the Poly-A RNA selection kit is required, whereas for total RNA-seq a pre-treatment with the RiboCop rRNA depletions is strongly advised to deplete the highly abundant ribosomal RNA from your sample, enabling more cost-effective sequencing.

When using a pre-treatment method, it needs to be taken into account that 100-200ng of total RNA will result in 1-2 ng poly-A enriched or rRNA depleted RNA.

When using total RNA without any depletion or enrichment, it is possible to work with inputs ranging from 100pg to 100ng.

Gene Detection
The high level of complexity of the CORALL libraries ensure faithful representation of the transcriptome, making this kit an excellent choice for sensitive expression profiling. The excellent gene discovery rates, Corall is delivering, do match the leading competitor kits.

figure 1: Gene detection. A. Gene discovery rates: the number of detected genes is plotted against the total number of reads mapping uniquely to exons B. Overlap of detected genes: The Venn diagram illustrates overlaps between CORALL and competitor kits

Transcript Coverage
Corall generates transcriptome-wide smooth and highly uniform read coverage

figure 2: Accumulated transcript body coverage for whole transcriptome: Coverage across all transcripts was generated using the tool provided by RSeQC

End-to-End coverage
CORALL’s improved and comprehensive coverage delivers best-in-class start and end site representation. Read coverage analysis, using the ERCC spike-in controls, clearly indicate that CORALL reads map more accurately to the exact ERCC TSS than competitor libraries that fail to cover the true start sites. In addition, CORALL provides superior coverage at the TES, compared to the competitor kits.

figure 3: Normalized ERCC coverage of A. (TSS) and B. (TES): Normalized coverage of accumulated mapped reads for all detected ERCC's. The absolute nucleotide positions relative to the TSS (red dotted line, A) and TES (blu dotted line, B) are shown

Unique Molecular Identifiers (UMI) seamlessly included
UMI’s are standardly included in the CORALL Library Prep kit and are seamlessly introduced into the libraries as an inherent part of the protocol. This results in Read 1 containing the UMI information, enabling direct access in the cost-efficient single-read mode.

Data Analysis
A Data Analysis Pipeline is now available on the BlueBee® Genomics Platform. The provided pipeline enables performing read quality control, mapping, UMI deduplication and transcript quantification.

SENSE mRNA-Seq Library Prep Kit v2

SENSE is a complete strand-specific mRNA-Seq Library Prep Kit for accurate gene expression profiling, transcriptome sequencing, discovery and quantification of antisense transcripts and overlapping genes. SENSE mRNA-seq Library Prep includes the Poly-A RNA selection kit (Cat. n° 039) enabling selective enrichment of mRNA.
o    >99,9% strand specificity
o    Fragmentation free library generation
o    1ng of total RNA input
o    Ready-to-sequence libraries in less then 5 hours
o    Unique Dual Indexing of up to 9.216 samples

Superior Strand Specificity
The strand-displacement stop/ligation technology used in the SENSE library prep kits generates fewer antisense artifacts which can be produced by template-switching in protocols which utilize RNA or cDNA fragmentation. This results in exceptional (99.9%) strand-specificity and reduced experimental noise, enabling the detection and quantification of antisense transcripts with high confidence.

Efficient rRNA removal
SENSE mRNA-Seq Library Prep includes the Poly-A RNA selection kit, which virtually eliminates cytoplasmic rRNA (0.001% of total reads from Universal Human Reference RNA) and removes the need for additional selection or depletion kits, saving you time and money

All-in-one solution
No additional kits or reagents for poly-A RNA selection, library amplification, size selection or purification or barcodes are required.

Different Sequencing Read Length
For good representation and even coverage of all transcripts, the library should have a size suitable for the chosen sequencing read length. The size of SENSE libraries can be adjusted, just by modulating the appropriate buffers during reverse transcription/ligation and purification steps.

Automation Friendly
To accommodate an automated setup, the SENSE mRNA kit is also available as autoSENSE mRNA-Seq v2 for Illumina. This solution accommodates easy automation on the Sciclone NGS Workstation. For other platforms, please contact us.

SENSE Total RNA-seq Library Prep Kit

The SENSE Total RNA-Seq Strand-Specific Library Prep Kit has been discontinued end of 2019 and is replaced by the CORALL Total RNA-Seq Library Prep kit  as the superior product.

Please contact us if you still have projects running on the SENSE Total RNA-Seq Library Prep Kit or in case you have questions.

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 the 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.

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

Dual Indexing (i5 indexing) Modules

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 colour 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 CORALL or SENSE library prep kits. Each of the i5 indices can be combined with any of the 96 i7 indices, included in the CORALL or SENSE kits, enabling up to 9.216 different barcoding options.
Compatible with:
•    CORALL Total RNA-Seq Library Prep Kits
•    SENSE mRNA-Seq Library Prep kit v2

i7 6nt Index Set (7001-7096)

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

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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 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.

Compatible with:
•    CORALL Total RNA-Seq Library Prep Kits
•    SENSE mRNA-Seq Library Prep kit v2

Ordering information:


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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.

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