Ovation® Rapid Library Systems
The Ovation® Rapid Library System provides the fastest NGS library construction workflow for producing libraries from as little as 100 ng genomic DNA in under 2 hours. The complete library kit is cost-effective, scalable and offers barcoding for multiplex sequencing.
This simple NGS workflow is suitable for a wide range of sequencing applications. The PCR-free workflow is particularly useful for applications sensitive to PCR bias or PCR artifacts such as sequencing of GC or AT rich regions of the human genome, or microbiome and prokaryotic samples.
Automation enabled on multiple platforms including Agilent Bravo, Beckman Biomek FXP, Perkin Elmer Sciclone, and Hamilton Star/Starlet.
Complete workflow in under 2 hours
- Simple add and incubate workflow without PCR amplification, eliminating PCR bias
- Quantitation-free and dilution-free workflow
- Libraries ready for single or paired-end sequencing
- Compatable with cDNA generated with the Ovation RNA-Seq System V2
Obtain uniform distribution of sequences in genome
- High reproducibility with no coverage bias
Unbiased libraries for extreme genomes
- PCR bias-free libraries with both low GC and high GC content genomes
Uniform distribution of multiplex sequencing reads
- Even distribution of barcodes among sequencing reads
- Digital Gene Expression (DGE)
- Amplicon Sequencing
|Compatible Platform||Illumina HiSeq, MiSeq, NextSeq|
|Starting Material||purified DNA, cDNA|
|Input Amounts||100– 500 ng|
The Ovation RNA-Seq System V2 (Part No. 7102) has been validated to work with the Ovation Rapid Library Systems.
Can I use FFPE or other degraded DNA as input into NuGEN DNA-Seq Library Systems?
We recommend using high quality DNA with the A260:A280 ratio in ecess of 1.8. Use of DNA samples with lower ratios may result in low library yield.
NuGEN does not provide the reagents used in the fragmentation steps. We suggest the Covaris instrument be utilized for DNA fragmentation, as suggested in the “materials” section of the User Guide.
I don’t have access to a Covaris instrument. Can I use alternative fragmentation methods?
We have only evaluated Covaris fragmented DNA during the development of the Ovation Rapid Library Systems. Other mechanical means of fragmentation, such as sonication, may be suitable.
RNAClean P beads are certified to be RNase and DNase free. We have tested both RNAClean P and AMPure P beads in our kits and observe no difference in performance between products.
What magnetic separation devices do you recommend for the SPRI bead purifications?
Due to the large number of commercially available magnets, we do not have a comprehensive list of compatible products. However, many magnets are compatible. As long as the magnet is strong enough to clear the solution of magnetic beads, it can be applied to the system. We have the following guidelines for selecting a magnetic separation device:
- Use a magnet designed for 0.2 mL tubes (PCR tubes), tube strips, or plates. Compared to magnets that are designed for 1.5 mL tubes, these minimize loss that can occur when samples are transferred from one tube to another.
- Prior to purchasing, check the manufacturer’s specifications for minimum and maimum volumes that can be effectively treated.
- Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the NuGEN workflow. This is also helpful to gain familiarity with the purification workflow.
How can I ensure maimum recovery of sample from the SPRI bead purification?
- Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
- Ensure that the beads are fully resuspended in solution before adding to the sample.
- Always use fresh ethanol during the washing steps. When preparing the ethanol, measure out the ethanol and water separately to ensure the desired ethanol concentration is obtained.
- Mi the bead suspension and sample thoroughly to ensure maimum binding of the samples to the beads.
Please refer to Quantitative and Qualitative Assessment of the Library in the User Guide for guidelines on quantitative and qualitative assessment. Libraries created with the Ovation Rapid Library System must be quantified using a qPCR based-method.
What is the epected yield of the library using the Ovation Rapid Library Systems?
The epected yield is >1000 pM (final library concentration), depending on the quality and quantity of the source cDNA or genomic DNA.
How many bases do the Ovation Rapid Library System adaptors add to the library?
The adaptors add 122 bp to the library.
Ovation Rapid Library Systems libraries are compatible with Illumina sequencing platforms.
What kind of sequencing primers can I use with your library?
The Ovation Rapid Library Systems are designed for use with the standard Illumina sequencing primers for both single end and paired-end sequencing applications.
Can the Ovation Rapid Library Systems be used with paired-end sequencing?
Yes, they can be used for both single end and paired-end sequencing. Special consideration should be given to the epected insert size in the paired-end assay. The epected distances between the 5´-most and 3´-most coordinates of paired-end reads will depend on the average fragment size of the insert pool.
How much material should I load into the sequencer?
Please follow manufacturer’s recommendations for library QC, quantitation, balancing and loading of the amplified library on the sequencer. For eperiments using the Ovation Rapid DR Multiple Systems 1–8 and 9–16, also see Appendi A. of the Ovation Rapid Library Systems User Guide.
What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?
For eperiments using the Ovation Rapid DR Multiple Systems 1–8 and 9–16 with dedicated read barcodes, please follow the Illumina recommendations on parsing barcodes. The NuGEN direct read barcodes are si-base unique barcode tags. The sequence of these NuGEN barcodes must be input prior to parsing.
How should I balance the libraries when preparing for a multipleed sequencing run?
For libraries produced using the Ovation Rapid DR Multiple Systems 1–8 and 9–16, refer to “Updated Low-ple Pooling Guidelines for NuGEN NGS Library Systems”, available on the NuGEN website.
We suggest that only perfect matches of all barcoded bases be considered for read binning. We recommend using specialized software for splitting barcoded reads into the appropriate groups. We have successfully used the following software tools for multiplex experiments:
Single End Parsing:
- Fastx Barcode splitter http://hannonlab.cshl.edu/fastx_toolkit/
- BARTAB http://www.biomedcentral.com/1471-2105’10/362
- Fastx-multx utility http://code.google.com/p/ea-utils/
- fastq-multx utility from ea-utils package http://code.google.com/p/ea-utils/
No. of Reactions
Ovation Rapid DR Multiplex System 1-8
Ovation Rapid DR Multiplex System 9-16
Ovation Rapid DR Multiplex System 1-96
For research use only. Not for use in diagnostic procedures.