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Ovation®
Ultralow System V2

The DNA-Seq Library Preparation Kit offering fast and scalable production of NGS libraries from degraded samples as low as 10 pg.
Overview
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Tab 01 / Overview
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Ovation® Ultralow System V2 features a proprietary DimerFree® technology that allows for efficient DNA-Seq library preparation

Next-generation sequencing (NGS) technology has been used in multiple research areas including cancer, medical, stem cell, and population genetics, where the starting input is often ultra-low or degraded DNA. When working with these difficult sample types, obtaining the maximum possible genomic information in a next-generation sequencing library poses a unique set of challenges. Standard DNA library preparation techniques can cause quality-related problems. Common issues such as low library yield and high percentages of reads derived from adaptor dimers limit the efficacy of the data generated.

 

Selection guide

Ovation Ultralow V2 DNA-Seq library preparation kit supports a broad range of inputs from human and non-human DNA – including DNA, cDNA generated from cell lines, fresh and FFPE tissue, liquid biopsy and cfDNA as well as microbial DNA. The DimerFree®  technology in this kit allows for efficient library preparation with virtually no adaptor dimers even at input levels as low as 10 pg.

Ovation® Ultralow System V2: The DNA-Seq library preparation kit offering several unique benefits:

  • Short, simple NGS library preparation workflow in as little as 3 hours with no hidden normalization or adaptor dilution steps
  • One kit for any sample type, including ultra-low input, intact or fragmented DNA
  • Works across a broad range of DNA inputs from 10 pg – 100 ng
  • Obtain a higher number of reads by eliminating adaptor dimers using our DimerFree technology
  • Ovation Ultralow System V2 is offered with single-index full length or Unique dual indexes (UDI) full length adaptors
  • Obtain broad coverage with high-fidelity amplification across a broad range of GC content
  • Easily automated on multiple liquid-handling platforms

Applications

  • Whole genome sequencing
  • DNA sequencing
  • ChIP sequencing
  • Targeted sequencing




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Tab 02 / Highlights
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Eliminate adaptor dimers

  • A higher percentage of informative reads compared to other kits
  • Cost effective and allows for a higher level of multiplexing
Show more

No detectable adaptor dimers.

1 ng gDNA created from Ultralow System V2 vs. Competitors X and Y analyzed on Agilent Bioanalyzer. Adaptor dimers are highlighted in the grey box.

A higher % of informative reads

Ovation® Ultralow System V2 results in the lowest amount of adaptor dimers produced, and 50% more informative reads than the competition at a 10 pg input.

Use a single workflow for any input amounts

  • Achieved high concordance between sample inputs ranging from 1 to 100 ng
  • Adaptor concentrations are pre-optimized, eliminating the labor-intensive adaptor dilution step


High concordance across a broad input range without the need to adjust adaptor concentrations. Scatterplots of the log FPKM and corresponding Pearson R values are shown for Ultralow Library Systems V2 libraries constructed from 1, 10 or 100 ng of double-stranded cDNA made from Human Brain reference RNA.

Obtain high quality results regardless of input.

Libraries were generated from 100 ng, 10 ng and 1 ng of human genomic DNA. Bioanalyzer analysis indicates no adaptor artifacts (grey box) regardless of input and without the need for adaptor dilution during library construction.

Directional libraries, simplified data analysis

Nucleotide Distributions of Forward (A) and Reverse (B) Reads. Maintaining the directional orientation of the original genomic DNA reduces the computational burden by a factor of two over non-directional libraries during Bismark data analysis. The forward read is always the C-to-T converted (original genomic) strand, while the reverse read is the G-to-A reverse complement.

Complete workflow in under 4 hours

  • Simple add and incubate workflow with minimum hands-on time
  • Faster time to data with better reproducibility and consistency


The Ovation® Ultralow System V2 provides a simple, robust single tube workflow that can be completed in 3-4 hours. Unique ligation chemistry results in clean, dimer-free libraries without the need for titrating adaptor concentration regardless of input quantity.

With RRBS there is no need to sequence the entire genome.

Top: Diagram of the Ovation RRBS Methyl-Seq kit final library structure. The location of the diversity bases and molecular tag (N6) are shown. Bottom:  Graphical representation of how unique molecules are identified based on the read alignment and molecular tag (N6) sequence.

Obtain results that are highly reproducible and concordant.

Concordance in methylation levels for CpG’s covered at 20x or greater depth between technical replicates using 25 ng of IMR90 gDNA (Left) and between Ovation RRBS Methyl-Seq System versus Whole Genome Bisulfite Sequencing (WGBS, Right).

Obtain broad coverage for DNA Sequencing

  • High fidelity PCR enzyme ensures unbiased libraries
  • Efficient libraries obtained from both AT-rich and GC-rich organisms


Even read distribution across a broad GC range. 1 ng of gDNA from E. coli (blue, 51% GC), R. sphaeroides (red, 69% GC) or S. aureus (green, 33% GC) were sequenced with Ultralow Library Systems V2. Experimental GC distribution (solid lines) closely matches theoretical distribution (dashed lines) from a GC of less than 20% to greater than 80%.




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

Illumina HiSeq, MiSeq, NextSeq, NovaSeq

Starting Material

Starting Material

Purified DNA, cDNA

Input Amounts

Input Amounts

10 pg – 100 ng




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Tab 04 / Faq
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FAQ

Input recommendations

Which Tecan 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) has been validated to work with this kit.

Can I use FFPE or other degraded DNA as input into Tecan DNA-Seq library prep kits?

We recommend using high quality DNA with minimal degradation. However, moderately degraded DNA may also work with these kits. The A260:A280 ratio for DNA samples should be in excess of 1.8 and A260:A230 should be in excess of 2.0. Use of DNA samples with lower ratios may result in low library yield or compromised results.

General workflow

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 sonication instruments or enzymatic fragmentation may be suitable but will need to be optimized.

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

Tecan 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 quantified independently before being pooled for use on the sequencer.

SPRI bead purifications

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:

  1. 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.
  2. Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
  3. Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the Tecan 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?

  1. Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
  2. Ensure that the beads are fully resuspended in solution before adding to the sample.
  3. 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.
  4. Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

Library quantification and qualification

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 Fragment Analyzer for the most accurate quantification.

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

The adaptors add 124 bp to the library.

Sequencing recommendations

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- and paired-end sequencing.

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

Yes, the kit can be used for both single- 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, quantification, balancing and loading of the amplified library on the sequencer.

Data analysis

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.




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Tab 05 / Literature
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Literature

Type
Title
No
User Guide
Ovation Ultralow Library Systems V2
M01379 V6.1
Application Note
M01348 V2.1
Product Sheet
Ovation Ultralow System V2
402666 V1.1
Safety Data Sheet
 

For research use only. Not for use in diagnostic procedures.
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