Swift Normalase™ KitsOnobird2019-06-20T16:33:40+00:00
Swift Normalase™ Kits
Revolutionary Library Normalization Technology for NGS Laboratories
The Swift Normalase Kit offers a novel enzymatic library normalization technology that is augmented by the power to consolidate library normalization and pooling for loading on Illumina® sequencing platforms. The Normalase workflow eliminates the need for library quantification and concentration adjustment prior to library pooling, resulting in optimal cluster density and library balance. The Swift normalization method can easily be integrated into standard protocols to improve turnaround time loading and accuracy for NGS laboratories.
Saves time and increases throughput: Uniform sample processing with fewer handling steps to generate balanced library representation in multiplexed sequencing
Reduces variability to save on sequencing costs: Improved library balancing allows higher multiplexing per run, predictable read depth, reducing the need to resequence.
Flexible design for many workflows: Compatible with diverse library preparation methods and sample types to produce more evenly balanced sequence data
Normalase is an enzymatic normalization of multiplexed next generation sequencing (NGS) libraries for equimolar pooling and balanced sample representation in sequencing.
What are the steps of the Normalase protocol?
The Normalase workflow consists of replacing conventional library amplification primers with Normalase primers and amplifying to a minimum yield threshold of 12 nM or more (20 uL volume), followed by Normalase I, library pooling, and Normalase II to generate 4 nM library pools. One bead-based cleanup post-amplification is used to remove oligonucleotides and small fragments.
What are the supported libraries for Swift Normalase Kits?
Libraries that have an amplified yield of 12 nM or higher (20 uL volume)
Libraries prepared for direct sequencing (e.g., whole genome, whole transcriptome)
Target enriched post-hybridization capture libraries that have indexed adapters
What insert sizes are appropriate to use with the Normalase protocol?
Best results are obtained using libraries with standard size distribution generated by Covaris or enzymatic fragmentation (i.e., 150-550 bp inserts) and bead-based size selection.
Libraries with broad or variable size distribution (i.e., transposase-based workflows) that demonstrate size dependent clustering effects that are independent of molarity may have 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 -20C and handled according to the protocol.
What is the library yield requirement to achieve performance with Normalase?
Achieving the minimum threshold of 12 nM in 20 µl is required for performance. Libraries that do not meet this threshold will be less than 4 nM post-Normalase and will be under-represented during cluster generation.
Are there specific recommendations for low-plex pooling?
Yes, If you are pooling less than 5 libraries or post-hybridization capture library pools, follow the recommendations provided in Appendix, Section A of Normalase protocol. If you require a higher final pool volume for sequencing on NovaSeq, 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 depth.
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 that can lead to higher variation in sample representation in sequencing data.
Also, 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 two weeks before sequencing. For longer term storage, safe stopping points are recommended following post-library amplification purification and post-Normalase I.
Is library amplification required?
Normalase does not necessarily introduce more PCR cycles but replaces the conventional primers in library amplification with Normalase primers, allowing the user to maintain their conventional library amplification workflow. If your conventional library preparation and amplification step yields ≥ 12 nM in 20 μl, simply replace conventional amplification primers with Normalase primers and run amplification with minimum adjustments to the final extension step. If prior to amplification your library preparation yields ≥ 12 nM, a minimum of 3 cycles using Normalase primers (Reagent R5) is required to condition the library for the downstream Normalase enzymology.
What causes variation of read counts (CV > 10%) between libraries within Normalase pools?
Sequencer overload, inconsistent pipetting of Normalase I Master Mix into individual libraries, or inconsistent pipetting of individual libraries into pools prior to Normalase II. Use P10 pipettor if available and ensure pipetting equipment is maintained and calibrated.
What if some of the libraries have a lower number of reads?
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.
What if recommended qPCR quantification of pools following Normalase II indicates yields below 4 nM?
If qPCR quantification indicated pools were below 4 nM, this may be due to a failure of a subset of samples within a pool. If this is observed, quantify your Normalase I-treated libraries to identify which libraries have failed or are < 12 nM.
Can I analyze the pools using fluorometric methods such as Bioanalyzer or Qubit?
Normalase produces single stranded libraries that are not measurable via Qubit or Bioanalyzer. Quantify via qPCR or run Bioanalyzer traces post-library amplification.