Description

CORALL mRNA-Seq Library Prep Kit

SARS-CoV-2_Icon   This product can be used for SARS-CoV-2 research.

The CORALL mRNA-Seq Library Prep Kit enables fast and cost-efficient generation of stranded, UMI labelled, and unique dual indexed libraries for whole transcriptome poly(A) RNA analyses using Illumina® NGS platforms.

Gene Detection

CORALL delivers excellent gene discovery rates across a wide range of RNA input amounts (Fig. 1).

Gene detection rate CORALL mRNA

Figure 1 | Gene discovery rates. 100 ng, 10 ng, and 1 ng Universal Human Reference RNA (UHRR) were used as input for poly(A) selection and library preparation using CORALL mRNA-Seq. Libraries were sequenced on Illumina® NextSeq500 (1×150 bp, 11.2 M reads / sample). The number of detected genes is plotted against the number of reads mapping uniquely to exons (calculated with featureCounts).

Transcript Coverage

CORALL generates transcriptome-wide smooth, uniform read coverage, comparable to that of competitor kits (Fig. 2).

CORALL_Figure_01

Figure 2 | Accumulated transcript body coverage (whole transcriptome). Coverage across all transcripts was generated using the geneBody_coverage.py tool provided by RSeQC (transcripts length normalized to 100 %).

Superior End-to-End Coverage

CORALL’s comprehensive coverage delivers improved transcript start and end site representation. Read coverage was analyzed using the ERCC spike-in controls, which feature precise, known transcription start and end sites (TSS and TES, respectively). CORALL reads map more accurately to the exact ERCC TSS (Fig. 3A) than competitor libraries, which fail to cover the true start sites. Additionally, CORALL provides elevated coverage at TES (Fig. 3B).

Figure 3 | Normalized ERCC coverage of A) TSS and B) TES. Normalized coverage of accumulated mapped reads for all detected ERCCs. The absolute nucleotide positions relative to the TSS (red dotted line, A) and TES (blue dotted line, B) are shown.

Flexible Input

CORALL mRNA-Seq Kits are the optimal choice for whole transcriptome mRNA sequencing. The kits include Poly(A) Selection for mRNA-enrichment prior to CORALL library generation and Lexogen’s UDI 12 nt Unique Dual Index Sets for amplification of unique dual indexed libraries. CORALL mRNA-Seq is compatible with total RNA inputs from 1 ng – 1 µg. High quality RNA (RIN, RQN >8) is recommended. CORALL libraries can also be prepared from rRNA-depleted RNA, e.g., using Lexogen’s RiboCop rRNA Depletion Kits. For samples with RNA integrity or quality scores (RIN, RQN) below 8, the CORALL Total RNA-Seq workflow with rRNA-depletion is recommended instead of CORALL mRNA-Seq.

Unique Molecular Identifiers (UMIs) seamlessly included

UMIs are seamlessly introduced into CORALL libraries allowing to remove PCR duplicates for accurate quantification. Read 1 contains the UMI information making it directly accessible 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 kit users to perform read quality control, mapping, Unique Molecular Identifier (UMI) deduplication, and transcript quantification. For using your activation code register an account with BlueBee (https://lexogen.bluebee.com/portal) and upload your data (fastq.gz files).

NOTE! BlueBee Data Analysis is available for a range of species, please refer to FAQ 7. Reference genomes for new species can be added upon request. Please note this will incur a fee. See CORALL Data Analysis, for further details.

Enhanced Multiplexing

CORALL mRNA-Seq Kits are available with up to 384 pre-mixed Unique Dual Indices (UDIs) for Forward Strand (A) and Reverse Complement (B) workflow (Set A1-A4: Cat. No. 158-161, 163, or Set B1: Cat. No. 162). Lexogen’s new 12 nt UDIs feature superior error correction for maximal sequencing data output and are also available as stand-alone Add-on Kits (Cat. No. 107 – 111) for use with other library preps. For further questions, please contact support@lexogen.com.

Fast and Easy All-in-One Workflow

CORALL mRNA-Seq Kits provide a complete solution for mRNA sequencing. RNA is poly(A) enriched in 1.1 hours and can be directly transferred into CORALL library preps. The streamlined RNA fragmentation-free protocol generates ready-to-sequence libraries in 4.5 hours. The complete CORALL mRNA-Seq workflow takes less than 6 hours.

“Transcriptional profiling is one of our key approaches to study and understand the molecular mechanism of action of cancer drugs. We have made excellent experiences with the CORALL kit both in terms of experimental feasibility but also data quality. Through integration with synthetic spike-ins SIRVs, It was particularly helpful when testing drugs that affect overall transcriptional output and thus cause a global decrease in mRNA levels.”

Georg Winter, PhD, Principal Investigator, CeMM, the Research Center for Molecular Medicine of the Austrian Academy of Sciences

Workflow

Poly(A) Selection
Step 1:
1 ng – 1 µg total RNA can be used for poly(A) selection.
Poly(A) RNA is enriched by hybridization to oligo(dT)
magnetic beads. Any RNA without poly(A) stretches
is washed away and poly(A) RNA is finally eluted.
03polya_workflow
CORALL Library Generation: RNA Input
Step 2:
After elution, poly(A) RNA can directly be used as template
for CORALL reverse transcription. No prior RNA fragmentation
is necessary, insert size is determined during reverse transcription.
CORALL Library Generation: Reverse Transcription
Step 2:
CORALL library generation is initiated by random hybridization
of Displacement Stop Primers (DSP) with partial Illumina-compatible
P7 sequences, to the RNA template. Reverse transcription extends
each DSP to the next one, where transcription is effectively stopped.
This stop prevents spurious second strand synthesis, maintaining
excellent strand specificity.
CORALL Library Generation: Linker Ligation
Step 2:
Highly efficient ligation of Linker Oligos to the 3’ ends of first-strand
cDNA fragments then introduces partial Illumina-compatible P5
sequences and Unique Molecular Identifiers (UMIs).
02c_corall-mRNA-workflow
CORALL Library Amplification: PCR
Step 3:
During PCR, second strand synthesis is performed,
and the double-stranded cDNA is amplified. In doing so,
unique dual i7 and i5 indices as well as complete adapter
sequences required for cluster generation on Illumina
instruments are added.
Sequencing
Step 4:
The UMI is positioned to be read out at the beginning of Read 1
during sequencing and can therefore be assessed even in Single-Read
mode. All purification steps are based on magnetic beads, rendering
the complete workflow highly suitable for automation.

FAQ

Frequently Asked Questions

Please find a list of the most frequently asked questions below. If you cannot find the answer to your question here or want to know more about our products, please contact support@lexogen.com.

CORALL has broad species compatibility and will work for all RNA, regardless of origin, as long as there is enough input material. CORALL mRNA-Seq (Cat. No. 158 – 163) can be used after poly(A) selection for mRNA-Seq of all poly(A) containing species. If ribosomal depletion is desired prior to CORALL, RiboCop is available for human, mouse, rat (Cat. No. 144, or Cat. No. 145 including globin depletion) as well as bacteria (Cat. No. 125 – 127). If you have further questions about species compatibility, please contact technical support at support@lexogen.com.

Any input amount between 1 ng and 1 µg of total RNA for rRNA depletion or poly(A) enrichment, or up to 100 ng input for total RNA without enrichment/depletion can be used for CORALL RNA-Seq.

High quality RNA (RIN >8) is required as input for poly(A) selection when using CORALL mRNA-Seq. For samples with RNA integrity or quality scores (RIN, RQN) below 8, we strongly recommend using rRNA depletion (Lexogen’s RiboCop rRNA Depletion Kits).

We do not recommend multiplexing Lexogen libraries with libraries from other vendors in the same sequencing lane.

Though this is possible in principle, specific optimization of index combinations, library pooling conditions, and loading amounts may be required, even for advanced users. Sequencing complex pools that include different library types at different lane shares may have unpredictable effects on sequencing run metrics, read quality, read outputs, and / or demultiplexing performance. Lexogen assumes no responsibility for the altered performance of Lexogen libraries sequenced in combination with external library types in the same lane (or run).

Due to size differences, libraries prepared with the Lexogen Small RNA-Seq Library Prep Kit (or any other small RNA library prep kit) should not be sequenced together with QuantSeq, QuantSeq-Flex, SENSE, or CORALL libraries. Please refer to the sequencing guidelines for each library type (library adapter details, loading amounts to use, and use of custom sequencing primers, etc), which are provided in our Library Prep Kit User Guides, and online Frequently Asked Questions (FAQs).

CORALL libraries can be sequenced either with single-read (SR) or paired-end (PE) sequencing. We can recommend SR50 – 200, or PE100 – 150. The choice of sequencing format may also depend on the particular application.

Please follow loading amount recommendations provided by Ilumina for your specific instrument, or contact info@lexogen.com for further information.

CORALL libraries can be analyzed using most standard RNA-Seq pipelines. The ideal workflow may depend on the goal of the sequencing experiment. Further information on recommendations for trimming, quality control, and alignment of CORALL reads can be found in the Data Analysis section of the CORALL product page, or in the CORALL User Guide, Appendix H, p.34.

A pipeline is available on the BlueBee® Genomics Platform that performs read quality control, mapping, Unique Molecular Identifier (UMI) deduplication, and transcript quantification.
009_CORALL_Workflow-Data-Analysis_V0200
Figure 1 | CORALL Data Analysis Pipeline Workflow.

Yes! For a limited time, CORALL kits are supplied with a code to access the CORALL data analysis pipeline on the BlueBee® Genomics Platform. Each code contains an equal number of pipeline runs as reactions provided in the kits.

ATTENTION: Data Analysis access is only free using the provided code, for fastq input files up to 5 GB in size. Larger fastq file sizes can only be processed using activation codes for large file sizes, which can be purchased additionally.

The analysis pipeline for CORALL is currently available for the following species:

Common name (if available) Species
African Oil Palm Elaeis guineensis
Arabidopsis Arabidopsis halleri
Arabidopsis Thale cress Arabidopsis thaliana
Baker’s Yeast Saccharomyces cerevisiae
Barley Hordeum vulgare
Bartonella Bartonella henselae
Brain-Eating Amoeba Naegleria fowleri
Chicken Gallus gallus
CHO-K1 Cell Line Cricetulus griseus
Common Yellow Monkeyflower Mimulus guttatus
COVID19 SARS-CoV-2
Cow Bos taurus
Cacao Tree Theobroma cacao criollo
Dog Canis lupus familiaris
Drummond’s Rockcress Boechera stricta
Ferret Mustela putorius furo
Fruit Fly Drosophila melanogaster
Fungus Fusarium oxysporum
Fungus Yarrowia lipolytica
Goat Capra hircus
Human Homo sapiens
Maize/Corn Zea mays
Melon Fly Bactrocera cucurbitae
Mouse Mus musculus
Nematode Roundworm Caenorhabditis elegans
Painted Turtle Chrysemys picta bellii
Pig Sus scrofa
Potato Solanum tuberosum
Pseudomonas Pseudomonas aeruginosa PA103
Purple False Brome Brachypodium distachyon
Rabbit Oryctolagus cuniculus
Rat Rattus norvegicus
Rice Oryza sativa
Salmon Salmon salar
Sorghum Sorghum bicolor
Sponge Amphimedon queenslandica
Starlet Sea Anemone Nematostella vectensis
Tomato Solanum lycopersicum
Water flea Daphnia pulex
Western Balsam Poplar Tree Populus trichocarpa
Yeast Candida albicans, Candida auris, Candida parapsilosis
Yeast Kluyveromyces lactis
Zebrafish Danio rerio

.
New species pipelines can be added upon request for an additional fee. Please contact info@lexogen.com if your species of interest is not listed, and for pricing information.

Activation codes for data analysis can be purchased via the web shop. Each code enables 24 data analysis runs (i.e., 24 Fastq.gz files may be analyzed). Reference genomes for various species are available (please contact support@lexogen.com for more information).

For using your activation code register an account with BlueBee (https://lexogen.bluebee.com/portal) and upload your data (fastq.gz files) to a new project. To execute runs, navigate to your project and go to runs -> add run select the data you wish to analyze and the species from the dropdown list then click run.

The qPCR assay is recommended to set the optimal number of PCR cycles for the library amplification. This will avoid under or overcycling. Undercycling may result in insufficient library for sequencing, and overcycling can lead to higher duplication levels and quantification biases.

To perform the qPCR assay please ensure you purchase these additional reagents along with your CORALL Total RNA-Seq Library Prep Kit:

  • PCR Add-on Kit for Illumina (Cat. No. 020.96) – Available from Lexogen
  • SYBR Green I Nucleic Acid Stain (10,000x in DMSO) – Available from Sigma- (Cat. No. S9430) or ThermoFisher (Cat. No. S7585).

NOTE! The use of SYBR Green-containing qPCR Mastermixes is explicitly NOT recommended. The SYBR concentrations may be inhibitory, or the buffer/enzymes included may alter the qPCR efficiency. This may lead to inhibition of amplification in general, or inaccurate endpoint cycle number calculations.

Please note that the use of EvaGreen dye at final concentrations of 0.1x – 1x is not officially recommended, as it is not fully compatible with the specified qPCR assay for cycle number calculation. Amplification curves generated using EvaGreen may be delayed and show reduced maximal fluorescence intensity compared to SYBR Green I dye. Therefore, endpoint cycle numbers may need to be calculated differently and verified by Endpoint PCR testing.

The qPCR is particularly important for FFPE and low or variable quality RNA input samples, and low RNA input amounts. Please be aware that different sample types (species, tissues, cell types), or the use of different upstream RNA processing steps (i.e. enrichment and selection methods) may affect the mRNA or non rRNA content of the sample. Therefore, more or less PCR cycles may be required, even if input amounts between sample types are consistent.

Yes, low quality and FFPE samples can be used with CORALL. Protocol modifications are recommended for library preparation from degraded RNA and FFPE samples with CORALL library prep kit. The following recommendations have been evaluated with 25 – 500 ng total RNA input:

  • Perform DNase I treatment prior to rRNA depletion (without an intermediate cleanup step).
  • In step 32 use 36 µl of PB (instead of 27 µl) for single indexing, or 42 µl of PB (instead of 31.5 µl) for dual indexing.
  • Optional: Add SIRV-Set 3 (0.1 – 0.2 % of target RNA fraction) prior to DNase I treatment.

Sequencing is recommended with SR75.

For further questions, please contact support@lexogen.com.

Lexogen UDI 12 nt Unique Dual Indexing Add-on Kits are compatible with QuantSeq FWD and REV (Cat. No. 015, 016), QuantSeq-Flex (Cat. No. 033, 034, 035), CORALL (Cat. No. 095, 146) and SENSE mRNA V2 (Cat. No. 001) Library prep kits.

The Lexogen UDI 12 nt Unique Dual Indexing Sets are not compatible with Small RNA library preparation kit (Cat. No. 052).

First time user of CORALL?

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Please choose which CORALL RNA-Seq Kit you are interested in (required):
CORALL Total RNA-Seq with Single Indexing (Cat. No. 095)CORALL Total RNA-Seq with Unique Dual Indexing – Set A1 (Cat. No. 117)CORALL Total RNA-Seq with Unique Dual Indexing – Set B1 (Cat. No. 118)CORALL mRNA-Seq with Unique Dual Indexing – Set A1 (Cat. No. 158)CORALL mRNA-Seq with Unique Dual Indexing – Set B1 (Cat. No. 162)I am not sure which kit to use, please advise me.

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