Swift Normalase™ KitsOnobird2019-11-10T20:47:11+00:00
Swift Normalase™ Kits
Revolutionary Normalization Technology for NGS Libraries
Now compatible with indexing by PCR and indexing by ligation for both DNA and RNA library prep workflows
The Swift Normalase Kit is a novel enzymatic NGS library normalization technology that streamlines library balancing and pooling for ease of loading on Illumina® sequencing platforms.
The Normalase workflow eliminates the need for using library quantification and insert size determination to individually adjust concentrations prior to library pooling, enabling equal volume pooling for mid- to high-throughput sequencing labs.
This fast, robust and automatable workflow results in optimal cluster density and library balance and can easily be integrated into your standard DNA and RNA library preparation protocols to improve turnaround time, increase efficiency and reduce cost for NGS laboratories.
Save time and increase throughput: Uniform sample processing with fewer handling steps to generate balanced library representation for multiplexed sequencing.
Reduce sequencing costs: Improve library balancing, allow higher multiplexing per run and obtain predictable read numbers.
Flexible design: Compatible with diverse DNA and RNA library preparation methods to produce more evenly balanced sequence data.
Normalase is an enzymatic normalization tool for multiplexing next generation sequencing (NGS) libraries with equimolar pooling resulting in balanced sample representation on Illumina sequencing runs.
What are the steps of the Normalase protocol?
The Normalase workflow consists of library amplification with Normalase primers to a minimum yield threshold of ≥ 12 nM (20 µL volume), followed by enzymatic selection, library pooling, and enzymatic normalization to generate 4 nM library pools.
What are the supported library preparation workflows for Swift Normalase Kits?
Swift Normalase Kits can be applied to:
Libraries with full-length indexed adapters
Libraries with truncated adapters and indexing PCR (requires Cat. No. 68096)
Libraries that have an amplified yield of 12 nM or higher in 20 µL volume
Libraries prepared for direct sequencing (e.g., whole genome, whole transcriptome)
Libraries or pools of libraries target-enriched by hybridization capture
Is library amplification required?
Yes, libraries must be amplified and conditioned with Normalase primers. More PCR cycles are not necessarily required, but each library must be ≥ 12 nM following the post-Normalase PCR bead clean-up. Normalase allows the user to maintain their conventional library amplification reagents, with the exception of amplification primers.
For indexing by ligation and using Reagent R5 during Normalase PCR:
If your conventional library preparation and amplification step yields > 12 nM in the 20 µL final eluate, simply replace conventional amplification primers with Normalase primers and run the amplification with minimum adjustments to the final extension step. If prior to amplification your library yields are ≥ 12 nM, a minimum of 3 cycles using Normalase primers, Reagent R5, is required to condition the library for the downstream Normalase enzymology.
For indexing by Normalase PCR:
Amplification, indexing and conditioning with Normalase Combinatorial Dual Indexing primers and Reagent R6 will require more cycles compared to conventional indexing primers. See Normalase protocol and the specific Swift library preparation protocols for minimum cycle recommendations.
Do I need to quantify libraries after amplification and before Normalase I?
No, it is not necessary to quantify libraries before Normalase I if the libraries are all known to be > 12 nM. For example, if your library preparation and your standard nucleic acid sample inputs yield 30 nM consistently, quantification can be skipped. Quantification is only required if library yields are unknown and potentially under the 12 nM minimum threshold. For quality control purposes, Swift recommends quantifying libraries after Normalase PCR using a validated qPCR assay or fluorometric quantification assay.
Does Normalase replace post-PCR library quantification?
No, Normalase replaces the steps of variable volume library pooling or library concentration adjustment before pooling. Normalase removes the need to use relative quants and fragment analysis to normalize and pool libraries based on calculated molarities.
Are there any safe stopping points in the Normalase protocol?
Yes, safe stopping points are 1) following Normalase PCR and bead-based clean-up, libraries eluted in low EDTA TE can be stored at -20 °C, 2) following Normalase I incubation, libraries can be stored at -20 °C.
What HiFi polymerases are compatible with Normalase PCR?
The Swift Normalase Kit has been tested by Swift with the following high-fidelity polymerases:
Swift Hot Start High Fidelity Polymerase
KAPA HiFi HotStart ReadyMix
NEBNext Ultra II Q5® Master Mix
Our customers have had success with several other commercially available high-fidelity polymerases available on the market. Please contact [email protected]to inquire about your preferred high-fidelity polymerase.
Can I use my own library amplification primers or indexing amplification primers for Normalase PCR?
No, Normalase PCR requires replacing your amplification primers to condition your libraries for the downstream enzymology. Reagent R5 replaces standard library amplification primers for those libraries indexed by adapter ligation, and Reagent R6 along with two Normalase Indexing primer combinations replace your indexing primers for those libraries indexed by PCR.
What insert sizes are appropriate to use with the Normalase protocol?
Libraries with uniform size distributions generated by Covaris or enzymatic fragmentation (i.e., 200-350 bp inserts) and bead-based size selection. Best results are obtained by using libraries with similar median insert sizes as measured by fragment analysis. Libraries with broad or variable size distribution (i.e., transposase-based workflows) that demonstrate size dependent clustering effects on Illumina sequencers, which are independent of molarity, may have more variable results.
What is the shelf life of the Swift Normalase Kit?
The shelf life of the Swift Normalase Kit is at least 6 months from the time the product is delivered when stored at -20 °C and handled according to the protocol.
What is the library yield requirement to achieve expected performance with Normalase?
Achieving a minimum threshold of 12 nM in 20 µL volume after PCR is required for expected performance. Libraries that do not meet this threshold will be less than 4 nM post-Normalase and will be under-represented during cluster generation. Additionally, if multiple libraries are under 12 nM to start, the final library pool will be < 4 nM, resulting in potential underclustering of the pool on the flow cell.
Are there specific recommendations for alternative pooling strategies?
Yes, follow the recommendations provided in Appendix, Section A of Normalase protocol for low-plex pooling. If you require a higher final pool volume for sequencing on the NovaSeq, for example, follow the recommendations provided in Appendix, Section B.
Is there a minimum or maximum limit to the number of samples that can be placed into a single Normalase pool?
No; however, consider your desired number of reads for each sample and only pool those samples together that have the same required read numbers. Additionally, consider index compatibility as well as insert size. Pool libraries of comparable insert sizes that can be co-sequenced together to avoid size dependent clustering effects that are independent of molarity and can lead to higher variation in sample representation in sequencing data. Do not pool libraries with index combinations that have not been validated by the supplier or demultiplexing errors and loss of data may occur.
Does this protocol support library long-term storage?
Final pools post-Normalase inactivation contain single stranded DNA and can be stored at -20 ºC for up to four weeks before sequencing. For longer term storage, safe stopping points are recommended following post-library amplification purification and post-Normalase I.
What causes variation of read counts (CV > 10%) among libraries within Normalase pools?
Libraries not meeting the 12 nM minimum threshold, sequencer overloading and subsequent overclustering resulting in low sequencing quality, inconsistent pipetting of Normalase I Master Mix into individual libraries, or inconsistent pipetting of individual libraries into pools prior to Normalase II will all introduce more variability and increase variation of read counts to >10%. Ensure to achieve the minimum threshold of 12 nM in 20 µL library yield for optimal performance. Quantify problematic libraries prior to pooling to confirm suggested molarity. Use a P10 pipettor if available and ensure pipetting equipment is maintained and calibrated.
Can I analyze Normalase pools using fluorometric methods such as Bioanalyzer or Qubit?
No, Normalase produces single stranded libraries that are not measurable via fluorometric or fragment analysis. Quantify via qPCR or run Bioanalyzer post-library amplification to obtain library traces and yields for quality control purposes.