Ovation® Custom Target Enrichment System
My colleagues and I are using high-throughput sequencing methods to study age-related diseases and have employed the Ovation® Target Enrichment System for targeted sequencing and identification of genetic variants. In conjunction with this method, we have utilized the Ovation® Target Enrichment Data Processing Application for BaseSpace to perform data analysis. We found the application to be a valuable tool for eliminating bottlenecks associated with basic data processing. The application aids our studies by streamlining data analysis and accelerating the understanding of the functional impact of genetic variants on the underlying processes of age-related diseases.
The Ovation® Custom DNA Target Enrichment System enables the targeted interrogation of any genomic regions of interest including genes, variants, or intergenic regions. Starting with 10 –- 500 ng of good quality or FFPE-derived genomic DNA, this system utilizes Single Primer Enrichment Technology (SPET) for custom target enrichment to generate high quality data with excellent specificity (>80% bases on-target), minimal dropouts and even coverage. A single target enrichment workflow yields data that is amenable to analysis of variants (SNVs/SNPs, insertions and deletions) and also gene level copy number information (CNVs).
Sample types include purified RNA from cell lines, fresh frozen or FFPE tissues, clinical samples and tumor biopsies.
Automation enabled on the Perkin Elmer Sciclone NGS system.
Simple, complete workflow from 10 – 500 ng DNA
- Compatible with degraded or FFPE DNA samples down to 10 ng
- Integration of a molecular tag for unambiguous unique molecule identification
- Point and click sequencing data pre-processing available through BaseSpace
Outstanding data quality ensuring high-quality variant calls
- Uniform target region coverage across a range of input amounts with minimal dropouts
- Highly sensitive assay allows detection of low abundant SNPs
- Detection of gene level copy number variation
Easily customizable panels specific for your studies
- Innovative technology allows target enrichment from a set of single base regions to large regions over 20 Mb.
- Iterative design process allows for modifications and additions of genes and target regions.
- Various probe design styles allow for target enrichment specific to your sample type and desired analysis.
- Single nucleotide variants/polymorphisms (SNV/SNP)
- Inserts and deletions (InDels)
- Copy number variations (CNV)
|Compatible Platform||Illumina HiSeq, MiSeq, NextSeq, MiniSeq|
|Input Amounts||10 ng – 500 ng|
The custom design, target enrichment probes and all reagents necessary to create custom target-enriched, singleple or multiple libraries from genomic DNA ready for sequencing on the Illumina platform are included in the system.
Do I need any additional reagents or instruments?
Any laboratory that is set up for sequencing on the Illumina platform should have all the necessary reagents and labware to perform the library preparation and target enrichment. (see section II.B in the User Guide for a list of additional equipment, reagents and labware).
What are the available kit sizes?
Custom kits are available in 32 or 96 reactions with the corresponding number of unique barcodes. The kits are also available as a single sample enrichment or a multiple sample enrichment.
Cell-free DNA might require an optimized probe design that addresses smaller fragments, similar to FFPE material. If there is sufficient plasma DNA as input, this may be possible, but we have not yet performed the eperiment.
Can the Ovation Target Enrichment System be used on single cell level?
Genomic DNA from a single cell would likely require a pre-amplification step prior to the library prep and enrichment. We have not yet performed this study.
We generally recommend Covaris focused acoustic shearing, and other methods that generate the appropriate size distribution of fragments (500 bp for intact DNA) are acceptable.
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 maximum 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.
- Mix the bead suspension and sample thoroughly to ensure maimum binding of the samples to the beads.
Please refer to section V.K. of the User Guide for guidelines on quantitative and qualitative assessment. We recommend using a qPCR based-method in combination with the Agilent Bioanalyzer or Tapestation for the most accurate quantification.
What library size should I use to calculate the library concentration?
For target enrichment libraries constructed from high quality DNA or FFPE DNA, use the average fragment size calculated by the Agilent Bioanalyzer or other methods of size determination.
How many bases do the Ovation Target Enrichment System adaptors add to the library?
The adaptors add 165 bp to the library.
Ovation Target Enrichment Systems libraries are compatible with Illumina sequencing platforms. Certain workflows with low-diversity samples may not be supported on all platforms. Please see the recommendations for your specific sequencer.
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.
What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?
Each eight-base barcode is a minimum edit distance of 3 from any other barcode. This means that a minimum of three edits (replacement, insertion, or deletion) must occur before one barcode becomes a different barcode. For further details on the barcode design strategy, please refer to Faircloth BC, Glenn TC (2012), Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels. PLoS ONE 7(8): e42543. doi:10.1371/journal.pone.0042543.
What kind of sequencing primers can I use with your libraries?
The Ovation Target Enrichment System is designed for use with the standard Illumina sequencing primers for both single-end and paired-end sequencing applications.
Can the system accommodate long reads?
Forward reads up to 150 bp should work without a problem. Longer reads can be performed but may etend into the probe.
Can I do paired end sequencing of an Ovation Custom Target Enrichment System library?
Yes. For most applications single end reads are sufficient and advised. You may want to perform paired end sequencing when looking specifically for rearrangements like inversions or translocations. However, due to the probe etension technology employed by the target enrichment system, the first 68 bases of reverse read sequence are probe- and linker-derived and may require trimming prior to alignment.
A member of the NuGEN Technical Services team will work with you ensure the correct information is provided for a custom design. RefSeq IDs or genomic coordinates will generally suffice. Please contact Technical Support for additional information.
What is the turnaround time of a custom probe design?
If the targets are provided in the appropriate format, it should take no more than a couple of days for the design to be complete. For non-human sequence this process may take longer in order for the correct genome sequence to be identified and added to the design pipeline. We estimate 3–4 weeks between obtaining customer approval on a finalized design and shipping the custom kit.
Can regions with pseudogenes be enriched?
Yes. Probes can be designed for regions that target the differences between genes and pseudogenes.
How does the probe design cover long target regions?
For larger target regions, probes are tiled within the target region in order to make sure that the entire target region is accurately represented.
No. of Reactions
Ovation® Custom Target Enrichment System 32 Reactions
Ovation® Custom Target Enrichment System 96 Reactions
For research use only. Not for use in diagnostic procedures.