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Rapid EZ DNA-Seq Library Preparation Kit

Fast and robust enzymatic PCR-free solution for DNA-seq library preparation in less than 2.5 hours.
Overview
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Tab 01 / Overview
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Rapid DNA sequencing library prep in less than 2.5 hours

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.

 

Selection guide

Benefits of Rapid EZ DNA-Seq library preparation kit:

  • Short add and incubate workflow that can be completed in 2.5 hours
  • Detect index hopping with unique dual indexes (UDI) adaptors
  • Eliminate adaptor dimers without adaptor titration regardless of sample input using the DimerFree technology

Applications

  • Whole genome sequencing
  • Whole genome metagenomics
  • Amplicon sequencing
  • PCR-free libraries




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Tab 02 / Highlights
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Simple and robust enzymatic PCR-free solution for DNA-Seq library preparation

  • Add and incubate workflow without PCR amplification, eliminating PCR bias
  • Flexible and robust fragmentation to generate insert sizes from 200-500 bp without optimization
  • No sample conditioning or adaptor dilution steps necessary


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.

Achieve complete measurements of methylation with oxBS.

 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.

TrueMethyl oxidative bisulfite conversion enables accurate measurements of methylation.

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.

Robust enzymatic fragmentation consistent across different inputs and replicates

  • Flexible and robust fragmentation to generate insert sizes from 200-500 bp without optimization
  • Fragmentation consistent across different sample types, inputs and GC content
Show more

Enzymatic fragmentation consistent across different inputs

Tecan's enzymatic fragmentation yielded consistent fragment size regardless of input amount (200 ng - 500 ng) with human gDNA samples

Fragmentation consistent across different bacteria input amounts

Consistent fragmentation was achieved from 3 blend bacterial sample inputs regardless of input amount (200 ng - 500 ng)

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).




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Tab 03 / Specifications
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Specifications

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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FAQ

Getting started

What materials are provided with Rapid EZ DNA-Seq?

Rapid EZ DNA-Seq includes all necessary buffers, primers and enzymes for library construction. SPRI purification beads are not included.

What equipment is required or will be useful?

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.

Can this system be used with other library preparation workflows?

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.

Input recommendations

What methods do you recommend for DNA isolation?

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.

Can I use phenol-chloroform based extractions for DNA isolation?

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.

Can I use Rapid EZ DNA-Seq with DNA from any organism?

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.

Do I need to use high-quality DNA?

This kit is designed for use with DNA samples of high molecular weight with little or no evidence of degradation.

General workflow

Is it necessary to fragment my DNA prior to Adaptor Ligation?

Yes.

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?

Library quantification by qPCR is required. Please refer to section V. F. for guidelines on quantitative and qualitative assessment.

How many bases do Rapid EZ DNA-Seq adaptors add to the library?

The adaptors add 136 bp to the library.

Sequencing recommendations

What sequencers are compatible with your libraries?

Rapid EZ DNA-Seq libraries are compatible with Illumina sequencing platforms.

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.

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

Rapid EZ DNA-Seq libraries are designed for use with the standard Illumina sequencing primers for both single-end and paired-end sequencing applications.

Can Rapid EZ DNA-Seq libraries be used with paired-end sequencing?

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.

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.

Are any special considerations needed for how I process Rapid EZ DNA-Seq libraries?

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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Literature

Type
Title
No
User Guide
Rapid EZ DNA-Seq
M01515 V1.1
Product Sheet
401546 V1
Safety Data Sheet
Rapid EZ DNA-Seq
 
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