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://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.

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.

The Lexogen i5 6 nt Dual Indexing Add-on Kits contain the indexed i5 adapters only. They can be used to introduce dual indexing for all Lexogen library prep kits that include the Lexogen i7 6 nt Indices (015, 016, 095, 001). The indices of the i7 and i5 6 nt Index Sets are 6 nucleotides long. The Lexogen UDI 12 nt Unique Dual Indexing Kits contain pre-mixed i5 and i7 adapters, each 12 nucleotides long.

When using the UDI 12 nt Sets for introduction of Unique Dual Indexing to libraries prepared with Lexogen kits that include the Lexogen i7 6 nt Indices (015, 016, 095, 001), the PCR (yellow) from the library prep kits has to be replaced by the Dual PCR Mix (purple) from the UDI 12 nt Add-on kits and the 12 nt UDIs are used instead of the i7 6 nt indices.

For convenience the QuantSeq FWD and CORALL kits are available as versions containing the UDI 12 nt Sets instead of the i7 6 nt Index Sets (113-115 and 117-119, respectively).

For more information on Lexogen Unique Dual Indexing solutions please visit www.lexogen.com/indexing/12nt-dual-indexing-kits/.

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, 096) 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).