Ovation® Fusion Panel Target Enrichment System V2
A lung adenocarcinoma tumor that was determined to have an ALK rearrangement by clinical SNaPshot Genotyping (multiplex PCR assay) was grown as a patient-derived xenograft model. Using NuGEN’s fusion detection workflow, the tumor was confirmed to have an EML4-ALK fusion after two passages in mice
The Ovation® Fusion Panel Target Enrichment System V2 is an RNA fusion panel that enables the simultaneously interrogation of known and novel gene fusions of 502 genes previously implicated in fusions based on the COSMIC database and recent publications. This system, utilizes Single Primer Enrichment Technology (SPET) for RNA to enrich for all exon - exon junctions in the targeted genes. Used together with the Ovation® cDNA Module (Part No. 9103) and dedicated gene fusion detection software, NuFuseD, this system provides a complete solution from total RNA to actionable RNA fusion results.
The system can be used to screen for gene fusion events in cancer discovery and diagnositcs from different 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 assay to detect and discover gene fusions
- Interrogate 502 genes known to be involved in gene fusions
Complete, seamless workflow from total RNA to actionable gene fusions
- Start with as little as 10 ng high quality total RNA
- Optimized data analysis pipeline available through BaseSpace or as a local installation
- Prioritized list of detected fusions for secondary validation studies
Easily customizable panels specific for your studies
- Innovative technology allows target enrichment from 10 genes to over 500 genes
- Iterative design to incorporate new information and targets
- Known fusion detection
- Novel fusion detection
- Alternative splicing
|Compatible Platform||Illumina HiSeq, MiSeq, NextSeq, MiniSeq|
|Starting Material||Total RNA|
|Input Amounts||10 ng – 200 ng|
The target enrichment probes and all reagents necessary to create target-enriched, multipleed libraries from RNA ready for sequencing on the Illumina platform are included in the system. The RNA must be converted to double-stranded cDNA using the Ovation cDNA Module for Target Enrichment.
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?
Standard kits are available as 8, 32 or 96 rn size with the corresponding number of unique barcodes. The kits are also available as a single sample enrichment or a multiple sample enrichment. Custom kits are available in 32 or 96 reactions.
Can the workflow be automated?
The Ovation Fusion Panel Target Enrichment System V2 features a simple add and incubate workflow, so it can be automated. Contact your NuGEN account manager for more details if you wish to automate the system.
We recommend a column-based method, including:
- Norgen Biotek Total RNA Purification Kit
- Zymo Research Quick-RNA™ Kits
- Arcturus PicoPure® RNA Isolation Kit
- Ambion PureLink® RNA Mini Kit
- Qiagen RNeasy Kits
Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.
Can I use TRIzol® or other phenol-chloroform based etractions for RNA isolation?
We do not recommend the use of TRIzol® or similar methods as any carry over of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.
Can I use carrier RNA during RNA isolation?
We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Technical Support for more information.
How well does RNA from FFPE material work in your system?
FFPE material can be used with this system. Because the RNA is modified and degraded, we suggest starting with a larger amount of input material. For more information regarding the use of FFPE RNA as input, contact NuGEN Technical Support.
Can the Ovation Fusion Panel Target Enrichment System V2 be used on single cell level?
RNA 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.
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.M. 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 FFPE RNA, use the average fragment size calculated by the Agilent Bioanalyzer or other methods of size determination. For target enrichment libraries constructed from high quality RNA, the average fragment size calculated by the Agilent Bioanalyzer may be inaccurate and can lead to underestimation of the library concentration. In this case, use a fragment size of 450 bp to calculate the library concentration.
How many bases do the Ovation Fusion Panel 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 Fusion Panel Target Enrichment System is designed for use with the standard Illumina sequencing primers for both single-end and paired-end sequencing applications.
Can the Ovation Fusion Panel Target Enrichment System V2 accommodate long reads?
Forward reads up to 150 bp should work without a problem when starting with high quality RNA. When starting with FFPE RNA, longer reads may etend into the probe and adaptor sequences.
Can I do paired end sequencing of an Ovation Fusion Panel Target Enrichment System V2 library?
Yes, we recommend performing paired end sequencing when looking for gene fusions. However, due to the probe etension technology employed by the target enrichment system, the first 58 bases of reverse read sequence are probe- and linker-derived. The first 18 bp of the reverse read must be trimmed to remove the linker sequence before data analysis.
No. of Reactions
Ovation® cDNA Module for Target Enrichment
8, 32, 96
8, 32, 96
Ovation® Fusion Panel Target Enrichment System V2
8, 32, 96
8, 32, 96
Ovation® Fusion Panel Target Enrichment System V2 (Singleplex Enrichment)
8, 32, 96
8, 32, 96
The Ovation cDNA Module for Target Enrichment is required to convert total RNA to cDNA before input into the Ovation Fusion Panel Target Enrichement System V2.
For research use only. Not for use in diagnostic procedures.