New updates to Primer sets
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REALLY™ includes Optional gDNA removal step
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Deplete human -and- mouse rRNA with REALLY
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New updates to Primer sets • REALLY™ includes Optional gDNA removal step • Deplete human -and- mouse rRNA with REALLY •
SRSLY KITs for NGS
Get SERIOUS about your libraries
The SRSLY NGS Library Prep Kit provides a simple and efficient ligation-based single-stranded DNA library preparation method that is engineered to produce complex libraries from low inputs and/or degraded DNA in about 2.5 hours. The SRSLY Kits offer three different bead purification options (small, moderate, and large fragment retention) because DNA templates come in all shapes and sizes.
The SRSLY Kits are available in a modular format that allows the user maximum flexibility in the choice of the base kit (based on input amount, type or fragment length), reaction volume, bead-purification, and the addition of Unique Molecular Index (UMI) or Unique Dual Index (UDI) primers used during PCR amplification.
Which “plus” kit should i get!
The SRSLY NGS Library Prep Kit is now available in four main formulations.
REALLY-rNONE™ is our latest offering for RNA-Seq with ribo-depletion, a single modular bundles work for 10ng - 250ng total RNA. We offer two versions of our standard SRSLY® kits (with or without a uracil-tolerant index PCR polymerase). For DNA template types that contain uracil, such as aDNA, methyl-converted DNA, and some FFPE samples, consider purchasing the kit supplied with the uracil-tolerant index PCR polymerase. For all other template types, the standard index PCR polymerase is the best choice. If utilizing the SRSLY UMI add-on reagents choose the standard polymerase option regardless of input type.
Visit our FAQs page or contact technicalsupport@claretbio.com for input-specific recommendations.
Modular components
follow the flowchart to choose from each module
Each base kit type is offered in 24- and 96-reaction format. The CBS-UD- primer sets (red) allow Unique Dual Indexing, but they are not compatible with the UMI addition option. For UMI addition choose the UM- primer sets (teal). Choosing the CBS-UM- primer sets also provide the primer and reagents for UMI addition, and primers for dual indexing PCR. If UMI addition is not required, the user may use their own Illumina® compatible index PCR primers by choosing “None”.
Refer to the “KIT CONTENTS NITTTY GRITTY” section below for more information about each module and subsequent sections for more information for UMI and UDI considerations
Assemble your modular kit in this order: First 1. Choose a base kit and 2. choose the number of reactions. Then based on the reaction volume 3. choose either the UDI or UMI primer strategy and 4. choose your beads (not for REALLY bundle though, beads are included!), finally 5. for gDNA (FFPE or fresh frozen) application choose the enzymatic fragmentation module.
KIT Contents nitty gritty
See below for more detailed description of kit components and available choices for every module
Please note: We have recently changed our tube caps
Please refer to Appendices A, B in the unabridged manual for more details on Index PCR primer set-up and UMI-aware sequencing set-up. Contact Illumina® technical support for more information about sequencer specific set-up for UMI-aware sequencing.
Please refer to this application note for comparison metrics between the Clarefy™ beads and other commercially available DNA purification beads. Please refer to Appendix C and the Beads Purification information section for bead selection strategies.
WHAT’S THE DEAL WITH UMI
Unique Molecular Index (UMI) addition is an optional feature of the SRSLY protocol. UMIs are molecular barcodes useful for detecting unique molecules, SNPs, and resolving PCR duplicates. The UMI addition module (UR- ) provides users a robust solution to add UMIs to any library prepared with the SRLSY Kit. The UMI addition step (performed prior to index PCR) tags a random 9 nt UMI to every SRSLY adapter ligated molecule via primer extension. The index PCR primers used subsequently (CBS-UM-24 or CBS-UM-96) only amplify the UMI-containing strand. If a user wishes to perform UMI-addition step, they must specifically choose this module. Please note that the UMI addition polymerase is indeed Uracil tolerant. Therefore if using the UMI addition module with SRSLY for samples with high deamination, index PCR polymerase substitution is not required. Using the regular UDI primers (CBS-UD-24 or CBS-UD-96 ) after UMI addition will erase the UMI information, please be cautious while choosing the primer-strategy.
There are pre-requisite considerations while setting up a UMI-aware sequencing run and UMI demultiplexing. We provide a description on how to set-up an UMI-aware sequencing run in the unabridged manual. This optional product is best-suited for experienced researchers. However, we offer an open-source bioinformatic solution to help customers demultiplex UMI-containing sequencing reads (Refer to our Software Page).
How unique is my udI
Unique Dual Indexes (UDI) uses unique or non-redundant i5 and i7 indexes for every library. This ensures unambiguous demultiplexing of reads after sequencing. UDIs also mitigates the effects of index-hopping observed with Illumina® sequencers with ExAmp chemistry. This is different from Combinatorial Dual Indexes (CDI) where i5 and i7 dual index combinations are unique, however each i5 or i7 may be repeated in a different library.
In the primer selections offered with SRSLY™ NGS Library Preparation Kits:
1. CBS-UD-24 primer set contains twenty four pairs of distinct i5 and i7 indexes (24 UDI), one reaction volume in each tube. Previously we offered only 12 UDI combinations (2X12 UDI), two reactions in each tube.
2. CBS-UM-24 primer set contains twelve unique i5 and i7 indexes, allowing 12 unique combinations of index pairs.
3. CBS-UD-96 primer plate one reaction each of ninety-six distinct i5 and i7 indexes (1X96 UDI)
4. CBS-UM-96 primer set contains ninety-six distinct indexes in the i7 Extension primer (used during UMI extension) and ninety-six distinct indexes in the i5 primer (provided as a primer pair with the Universal i7 primer, to be used during Index PCR). This allows 96 UDI combinations.
Please refer to Appendix A and B in our unabridged manual for more information about the indexes offered with SRSLY kits.
ENSURING A QUALITY PRODUCT
All the components in our SRSLY NGS Library Prep Kit for Illumina are functionally validated through NGS library construction. For every SRSLY kit lot manufactured we prepare SRSLY libraries from a control (as prepared in the Unabridged User Manual) and no-template controls. SRSLY libraries are quantified for yield and size distribution. All libraries (excluding the no-template controls) are then sequenced on an Illumina MiSeq platform. Libraries are then quantified for complexity as well as several other rigorous in-house metrics.
