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Fast and robust enzymatic PCR-free solution for DNA-seq library preparation in less than 2.5 hours.
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Tab 01 / Overview
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As NGS technology advances, the need for highly efficient, unbiased library generation for a wide variety of inputs has grown. High-throughput sequencing operations need library preparation workflows that optimize many factors, such as sample preparation and processing efficiency without compromising the quality of downstream bioinformatics analysis.
Rapid EZ DNA-Seq library preparation kit can meet these needs as it provides a fast and simple workflow with optimization-free, robust enzymatic fragmentation, and DimerFree® library construction.
The PCR-free workflow in Rapid EZ library preparation kit contains only one bead clean-up step and supports samples that are sensitive to PCR bias or PCR artifacts such as the sequencing of GC- or AT-rich regions of the human genome, or microbiome and prokaryotic samples.
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Tab 02 / Highlights
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Rapid EZ DNA-Seq library preparation - The streamlined workflow starts with enzymatic fragmentation of intact DNA followed by adaptor ligation and final repair to produce sequencing-ready libraries.
The schematic (Left) shows classic bisulfite conversion, which creates a library that detects both 5mC and 5hmC. Processing with the oxidation of 5hmC (Right) generates a bisulfite-convertible base that leads to detection of only 5mC. Differences between the libraries can then be used to deduce the sites of 5hmC modifications.
A) Standard bisulfite conversion cannot distinguish between 5mC and 5hmC, resulting in a single readout. B) TrueMethyl oxidative bisulfite conversion provides an accurate methylation profile of each.
Tecan's enzymatic fragmentation yielded consistent fragment size regardless of input amount (200 ng - 500 ng) with human gDNA samples
Consistent fragmentation was achieved from 3 blend bacterial sample inputs regardless of input amount (200 ng - 500 ng)
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.
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).
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Tab 03 / Specifications
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Specification |
Specification/Description |
---|---|
Compatible Platform |
Compatible Platform Illumina HiSeq, MiSeq, NextSeq, MiniSeq, NovaSeq |
Starting Material |
Starting Material purified DNA, cDNA |
Input Amounts |
Input Amounts 200 – 500 ng |
Workflow time |
Workflow time 2.5 hours |
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Tab 04 / Faq
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Rapid EZ DNA-Seq includes all necessary buffers, primers and enzymes for library construction. SPRI purification beads are not included.
Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, a magnetic plate for 0.2 mL tubes, strips, or plates and a fluorometer. An Agilent Bioanalyzer or Fragment Analyzer may also be useful for optional analytical tests. A comprehensive list of required and recommended equipment can be found in Section II.B of the User Guide.
Rapid EZ DNA-Seq is an end-to-end solution designed to generate libraries for Illumina sequencing starting from gDNA or cDNA and has not been tested with alternative library preparation systems.
We recommend a column-based extraction method, including: Qiagen QIAprep Miniprep or DNeasy Miniprep kits, Zymo Quick-DNA kits and Thermo Fisher PureLink Genomic DNA kits.
We do not recommend the use of these methods as any carryover of organics may inhibit downstream enzyme activity. If using, we recommend using a column-based purification of the downstream enzyme activity. If using, we recommend using a column-based purification of the DNA prior to input into the kit.
Rapid EZ DNA-Seq has been designed for use with a broad range of different organisms. Special consideration should be given when using low-input samples from organisms with large genomes.
This kit is designed for use with DNA samples of high molecular weight with little or no evidence of degradation.
Yes.
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.
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:
Library quantification by qPCR is required. Please refer to section V. F. for guidelines on quantitative and qualitative assessment.
The adaptors add 136 bp to the library.
Rapid EZ DNA-Seq libraries are compatible with Illumina sequencing platforms.
Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.
Rapid EZ DNA-Seq libraries are designed for use with the standard Illumina sequencing primers for both single-end and paired-end sequencing applications.
Yes. The libraries produced using this kit 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 workflow generates libraries with an average insert size that is dependent on the fragmentation conditions used.
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.
The final Rapid EZ libraries can be analyzed using standard pipelines. To remove adaptor artifacts use standard TruSeq sequences for read trimming.
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Tab 05 / Literature
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For research use only. Not for use in diagnostic procedures.