Description
CORALL Total RNA-Seq Library Prep Kit
The CORALL Total RNA-Seq Library Prep Kit enables fast and cost-efficient generation of UMI labelled, stranded libraries for whole transcriptome analyses using Illumina® NGS platforms.
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).
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 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). We recommend using Lexogen’s RiboCop rRNA Depletion Kit V1.2 (Cat. No. 037) or Poly(A) RNA Selection Kit (Cat. No. 039). 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.
Enhanced Multiplexing
CORALL libraries can be multiplexed using up to 96 i7 indices (included in the kit). 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 indices enable up to 9,216 different index combinations for sequencing.
Fast and Easy All-in-One Protocol
The streamlined RNA fragmentation-free protocol generates ready-to-sequence libraries in 4.5 hours.
Workflow
or rRNA depleted RNA as input.
of Displacement Stop Primers (DSP) with partial Illumina-compatible
P7 sequences, to the RNA template. No prior RNA fragmentation is
necessary, as the insert size is determined by the distance between
hybridized DSPs. Reverse transcription extends each DSP to the next,
where transcription is effectively stopped. This stop prevents spurious
second strand synthesis, maintaining excellent strand specificity.
cDNA fragments then introduces partial Illumina-compatible P5
sequences and Unique Molecular Identifiers (UMIs).
double-stranded cDNA is amplified. In doing so, i7 and (optional)
i5 indices as well as complete adapter sequences required for
cluster generation on Illumina instruments are added.
during sequencing and can therefore be assessed even in Single-Read
mode. All purification steps are based on magnetic beads, rendering
the protocol 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.
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.
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://www.bluebee.com/lexogen/) 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.
Downloads
CORALL Total RNA-Seq Library Prep Kit for Illumina
User Guide – update 24.09.2019
Product Flyer – update 20.09.2019
PCR Add-on Kit for Illumina Instruction Manual – update 03.07.2019
i5 Dual Indexing Add-on Kits (5001-5096) Instruction Manual – update 24.05.2018 (release of additional i5 Unique Dual Indexing Add-on Kit (5001-5096))
Lexogen i7 and i5 Index Sequences – including for kits bought before 17.02.2017
Library Quantification Calculation File
Material Safety Datasheets
MSDS information for CORALL Total RNA-Seq Library Prep Kit for Illumina
If you need more information about our products, please contact us through support@lexogen.com or directly under +43 1 345 1212-41.
CORALL Free Trial Kit
Dear Customer,
If you would like to receive a CORALL Total RNA-Seq Free Trial Kit, please fill in the form.
If you have any questions, please do not hesitate to contact us at info@lexogen.com or +43 (0) 1 345 1212 – 41.
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