The Purification Module contains all the necessary reagents to carry out additional purifications for example after PCR or to concentrate libraries if needed.
Compatible for QuantSeq, CORALL, Small RNA-Seq and TeloPrime.
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 QuantSeq 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.
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 color 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 QuantSeq 3’ mRNA-seq kits. Each of the i5 indices can be combined with any of the 96 i7 indices, included in the QuantSeq kits, enabling up to 9.216 different barcoding options.
The UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina) contains the UMI Second Strand Synthesis Mix (USS). This mix simply replaces the Second Strand Synthesis Mix 1 (SS1) from the standard QuantSeq FWD kit.
o Unique Molecular Identifiers (UMI) for detection and elimination of PCR duplicates
o Streamlined during QuantSeq FWD Library Preps
o Free data analysis pipeline on the Bluebee® Genomics Analysis platform
Use this module to identify PCR duplicates and eliminate amplification bias.
The Globin Block (RS-Globin Block) Modules for QuantSeq prevent the generation of library fragments from globin mRNA’s by blocking their extension during second strand synthesis. This module is intended for the preparation of libraries from blood RNA samples. The module is compatible only with QuantSeq 3’ mRNA-seq library prep kits for Illumina FWD (Cat. n° 015.xx) and REV (Cat. n° 016.xx)
Each module provides a modified RNA removal solution (RS-Globin block), containing species-specific globin blocking oligos. The RS-Globin block solution replaces the RNA removal solution from the standard QuantSeq 3’ mRNA-seq library prep kits. Separate modules are available for human (Homo sapiens) and pig (Sus scrofa)
The RS-Globin Block Homo sapiens module (RS-GBHs) is designed for human blood and contains blocking oligo’s against the human alpha en beta hemoglobin chain mRNA’s HBA1, HBA2 and HBB
The RS-Globin Block Sus scrofa module (RS-GBSs) is designed for pig blood and contains blocking oligo’s against the pig alpha and beta hemoglobin chain mRNA’s HBA and HBB.
The BC1 Block (RS-BC1 Block) Module for QuantSeq prevents the generation of library fragments from the abundant BC1 transcripts that are present in mouse brain samples (Mus musculus, Mm), by blocking their extension during second strand synthesis. The module is compatible with QuantSeq 3’ mRNA-Seq Library Prep Kits for Illumina (FWD, Cat. No. 015, 113 – 115, 129 – 131, and REV, Cat. No. 016), and is intended for the preparation of libraries from mouse brain samples.
The module provides a modified RNA Removal Solution (RS-BC1 Block) containing transcript-specific BC1 blocking oligos. The RS-BC1 Block solution replaces the RNA Removal Solution (RS) from the standard QuantSeq 3’ mRNA-Seq Library Prep Kits.
The QuantSeq-Flex Targeted RNA-Seq Modules complement the QuantSeq FWD kits and enable the generation of targeted RNA-Seq libraries from any RNA sample using custom primers for first or second strand synthesis.
With the QuantSeq-Flex First Strand Synthesis Module V2 the reverse transcription primer of QuantSeq FWD can be substituted with custom target-specific primers.
The new QuantSeq-Flex First Strand Synthesis Module V2 can further be used to modulate the insert size or to enable larger RNA volumes for standard QuantSeq FWD library preps.
With the QuantSeq-Flex Second Strand Synthesis Module V2 the random second strand synthesis primer can be substituted with a target-specific primer in the QuantSeq protocol. Please note that the second strand synthesis reaction requires annealing temperatures of >45 °C. In case you need lower temperatures, please contact us.
If dual-targeted RNA-Seq (custom primers in first and second strand synthesis) is required, please contact us to request an optimized product.
The Gel Extraction Module enables the size selection of Small RNA-Seq libraries in a convenient spin column format. It prevents unwanted sequences (e.g., linker-linker artifacts and longer library fragments) dominating the NGS results and helps focus the sequencing depth on the desired microRNA fraction.
The module includes a reagent box consisting of Loading Dye, NGS MW Ladder, and NGS Control Ladder, and a purification box with all necessary buffers and purification columns for clean-up.
Compatible for Small RNA-Seq kits
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.
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 ths 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.
1 When sequencing libraries indexed with UDI Sets A1-A4 on Illumina instruments that use the Reverse Complement Workflow for dual-indexing, the i5 index sequences must be entered in the reverse complement orientation on the sample sheet for demultiplexing. EXAMPLE: i512A_0002 should be entered as TTAGTAACTGGG instead of CCCAGTTACTAA in the sample sheet for demultiplexing. The reverse complement sequences (i5rc) are provided the index sequence sheet.
2 All 12 nucleotides of the i5 index must be read out for error correction using Lexogen’s Error Correction Tool and the index read should be analyzed as the reverse complement, in this case CCCAGTTACTAA from the example above (i512A_0002).
3 Loading 2 individual lanes on a NovaSeq Xp 2-Lane or 4-Lane Kit using the Xp Workflow.
4 Loading 3 individual lanes on a NovaSeq Xp 4-Lane Kit using the Xp Workflow.
5 Loading 4 individual lanes on a NovaSeq Xp 4-Lane Kit using the Xp Workflow.
Table 2 | Lexogen UDI 12 nt Unique Dual Indexing product compatibility with Library Prep Kits.
1 QuantSeq-Pool kits contain 96 inline barcodes (i1) for individual sample barcoding before pooling. For analysis of more than 96 samples additional indexing with e.g. UDIs is required.
2 LUTHOR kits don’t contain barcodes. Additional purchase is required for functionality of the library prep.
3 The amplification reagents which are provided in the QuantSeq-Rev and QuantSeq-Flex kits have to be replace by the amplification reagents provided in the UDI Add-on kits (as instructed in the UDI Add-on Kit Instruction Manual).
4 Libraries (RNA-Seq and DNA-Seq) from other vendors must contain TruSeq™ – compatible stubby adapters (where partial Illumina adapters are introduced during the workflow and completed with the index information during the endpoint PCR step). When unsure about compatibility please contact support@isogen-lifescience.com .
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support@isogen-lifescience.com
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