Transcript Coverage
CORALL generates transcriptome-wide smooth, uniform read coverage, comparable to that of competitor kits (Fig. 1).
Figure 1 | 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 %).
Gene Detection
CORALL delivers excellent gene discovery rates matching conventional competitor kits (Fig. 2A). 95% of genes are commonly detected between CORALL and each of the two competitors (at >10 counts per million (CPM), Fig. 2B). This high level of library complexity ensures faithful representation of the transcriptome, enabling sensitive expression profiling.

Figure 2 | Gene detection. A) Gene discovery rates. The number of detected genes is plotted against the total number of reads mapping uniquely to exons (calculated with featureCounts). B) Overlap of detected genes. The Venn diagram illustrates overlaps between CORALL and competitor kits for genes detected with normalized expression levels >10 CPM (for uniquely mapping reads).
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).
Flexible Input
CORALL libraries can be prepared from poly(A)-enriched or rRNA-depleted RNA (100 – 200 ng of total RNA yields approximately 1 – 2 ng of rRNA-depleted RNA). CORALL is available as a stand-alone kit with single indexing or UDIs (Cat. No. 095, or 117 – 119 and 132 – 134) or in a bundle with RiboCop rRNA Depletion Kits (Cat. No. 146, 147). For mRNA analysis we recommend to use the Poly(A) RNA Selection Kit (Cat. No. 039) upstream of CORALL. Total RNA without prior depletion or enrichment (100 pg to 100 ng) can also be used as input.
Unique Molecular Identifiers (UMIs) seamlessly included
UMIs are seamlessly introduced into CORALL libraries as an inherent part of the protocol. As a result 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 libraries can be multiplexed using up to 96 i7 indices (included in the single indexing kit, Cat. No. 095, 146). Additional i5 indices can also be introduced using the Lexogen i5 6 nt Dual Indexing Add-on Kits (Cat. No. 047). Used together, Lexogen’s 96 i7 and 96 i5 6 nt indices enable up to 9,216 different index combinations for sequencing.
NEW! CORALL Total RNA-Seq library prep is now also available with up to 384 pre-mixed Unique Dual Indices (Cat. No. 117-119, 132-134). UDIs are also included in CORALL mRNA-Seq Kits (Cat. No. 158-163). 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 Protocol
The streamlined RNA fragmentation-free protocol generates ready-to-sequence libraries in 4.5 hours.
“We have successfully used the CORALL kit in our latest project to generate high quality sequencing libraries from low input material. With its fast and clear protocol we were able to generate the libraries in less than one day. We will surely use the CORALL kit again and recommend it for whole transcriptome analysis.”
“We used the new CORALL kit for performing transcriptome-analysis of CRISPR-modified cells in order to understand the consequences of deregulated epigenetic modifiers. In our hands the kit performance was highly satisfying in terms of data-quality and reproducibility across biological replicates. It furthermore convinced us with the ease of use, clarity of instructions, details in the manual, and handling of reagents.”
Workflow
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).
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).
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.
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).
Downloads
CORALL Total RNA-Seq Library Prep Kit for Illumina
User Guide for CORALL Total RNA-Seq – update 31.03.2020
User Guide for CORALL RNA-Seq with UDIs – update 02.12.2020
Send us your publication & get the RNA T-shirt!
User Guide – CORALL RNA-Seq Integrated Data Analysis Pipelines on BlueBee® Genomics Platform – update 20.01.2021
Product Flyer – update 20.09.2019
PCR Add-on Kit for Illumina Instruction Manual – update 04.11.2020
i5 Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 12.03.2019
Lexogen i7 and i5 Index Sequences – update 05.05.2020
Library Quantification Calculation File
Material Safety Datasheets
MSDS Information can be found in the Documents page.
If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.
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