Ovation^® Ultralow Library System V2

Which NuGEN amplification system kits can be used to produce dsDNA for input to the Ovation Ultralow System V2?

The Ovation RNA-Seq System V2 (Part No. 7102) and Ovation RNA-Seq FFPE System (Part No. 7150) have been validated to work with the Ovation Ultralow System V2 1–96.

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 excess of 1.8. Use of DNA samples with lower ratios may result in low library yield.

I don’t have access to a Covaris instrument. Can I use alternative fragmentation methods?

We have evaluated only Covaris fragmented DNA during the development of the Ovation Ultralow System V2. Other mechanical means of fragmentation, such as sonication, may be suitable.

Does NuGEN provide reagents for performing the fragmentation step of the protocol?

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.

Can I modify the number of PCR amplification cycles recommended by the Ovation Ultralow System V2 workflow when using different DNA input amounts?

Generally speaking, fewer PCR cycles will be needed when working with larger input amounts. See Table 6 of the User Guide for guidelines on the number of cycles to use.

Can I combine the barcoded libraries prior to the PCR amplification step?

This is not recommended. The stoichiometry of barcoded libraries may be adversely affected by this modification to the workflow. We suggest that the libraries be amplified and quantitated independently before being pooled for use on the sequencer.

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP 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 maximum 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 maximum binding of the samples to the beads.

How do I measure my final library yield? Can I use an Agilent Bioanalyzer to evaluate the product?

Please refer to section V.L. 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.

How many bases do the Ovation Ultralow System V2 adaptors add to the library?

The adaptors add 122 bp to the library.

What sequencers are compatible with your libraries?

Ovation Ultralow Systems V2 libraries are compatible with Illumina sequencing platforms.

What kind of sequencing primers can I use with your library?

The Ovation Ultralow System V2 is designed for use with the standard Illumina sequencing primers for both single end and paired-end sequencing applications. Illumina sequencing primers are used for multiplex sequencing.

Can the Ovation Ultralow System V2 be used with paired-end sequencing?

Yes, the System can be used for both single end and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The expected 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.

What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?

Each 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.