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Celero™ EZ DNA-Seq library preparation workflow

Streamlined DNA-Seq library prep kit integrating enzymatic fragmentation for a 3 step workflow. Integrated with NuQuant library quantification to generate quantified libraries ready for sequencing.
Overview
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Tab 01 / Overview
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Celero Modular

In addition to our standard Celero products, we offer customizable Celero solutions for high throughput DNA-seq, high throughput amplicon sequencing, and 384 multiplexing with NuQuant technology.

Please speak with an NGS Expert to find out more.

DNA-Seq with enzymatic fragmentation in 3 simple steps

Celero EZ DNA-Seq provides an innovative solution to streamline whole genome sequencing (WGS) library preparation and quantification. This library prep kit features a fast, easy-to-use, addition-only workflow that eliminates post-ligation bead purification, resulting in faster library preparation and reduced hands-on time.

 

Selection guide

Celero EZ DNA-Seq features our enzymatic fragmentation technology that is simple, robust and consistent for fragmentation of genomic DNA into homologous sized fragments. This technology provides greatly improved fragmentation without optimization, outperforming standard DNA-Seq kits for a broad range of sample types and concentrations to provide the best possible starting material for your sequencing protocol.

DNA libraries from Celero EZ provide higher yields and better uniformity of coverage, ideal for downstream target enrichment applications like whole exome sequencing. When combined with Tecan’s DreamPrep™ NGS automation workflow, Celero EZ DNA-seq delivers a robust solution for high throughput WGS and target enrichment applications.

  • Broad input range from 10 ng – 500 ng
  • Tunable, optimization-free enzymatic fragmentation for all standard sample types
  • No adaptor or template dilution and no post-ligation purification step leading to reduced hands-on time
  • Flexible multiplexing with 384 dedicated dual index or Unique Dual Index (UDI)† adaptors
  • Integrated library quantification to simplify library pooling

This library preparation kit is integrated with NuQuant®†, a proprietary library quantification method for efficient and accurate quantitation of Next-Generation Sequencing (NGS) libraries. The NuQuant method eliminates the need for time-consuming or inaccurate library quantification methods like qPCR, fluorometry (e.g. Qubit®) or microfluidic electrophoresis (e.g. Bioanalyzer®) allowing DNA-Seq libraries to be constructed and quantified in less than 3 hours.

Applications

  • Whole genome sequencing
  • Exome sequencing
  • Microbiome




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Tab 02 / Highlights
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A simple and robust solution for DNA-Seq library preparation

  • A single workflow for DNA-Seq library preparation with integrated library quantification method
  • One bead clean-up step following PCR
  • Flexible and robust fragmentation to generate insert sized from 200-500 bp
  • No sample conditioning or adaptor dilution steps necessary
Show more

Celero EZ DNA-Seq has a shorter workflow compared competitor workflows

Comparing DNA-Seq library preparation workflow of Celero Enzymatic with competitive kits. Celero has the least steps and is integrated with NuQuant library quantification method.

Celero EZ DNA-Seq has a simple, easily automatable workflow

Celero Enzymatic Fragmentation with PCR workflow offers a simple and quick workflow for generating sequencing-ready quantified libraries.

NuQuant Library Quantification Method: Accurately quantify molar concentrations of NGS libraries

NuQuant library quantification is a proprietary method by which fluorescent labels are incorporated into the library molecules during the library preparation process. Each library molecule has an equivalent number of labels incorporated, regardless of the size of the library fragment, resulting in a direct measurement of molar concentration using standard fluorometers. NGS library quantification with NuQuant is accurate, easy to use and is integrated with the Celero PCR Workflow with Enzymatic Fragmentation DNA-Seq library preparation kit.

  • Accurately measure molar library concentration without the need for library size analysis (e.g. Bioanlyzer) unlike Qubit mass measurement or qPCR
  • Quantify only sequenceable molecules unlike Qubit and Bioanalyzer
  • Eliminate the need of time-consuming qPCR for library quantification
  • Compatible with standard fluorometers such as the Qubit or standard plate readers
Show more

NuQuant is more accurate than traditional methods

Library quantification with the NuQuant method is more accurate than Bioanalyzer and qPCR.

NuQuant saves time and money compared to traditional quantification methods

NGS library quantification with NuQuant compared to Qubit, qPCR and Bioanalyzer.

Whole exome sequencing with Celero EZ DNA-Seq

  • Enable more accurate variant calling - Celero EZ DNA-Seq library preparation kit consistently maintains greater than 98% target coverage
  • Even depth of coverage of target sequences compared to competitor
  • Greater uniformity of coverage reduces the number of reads required, enabling further cost savings 
Show more

Celero EZ is a streamlined library preparation solution with one-bead clean up step compared to traditional workflows enabling a more efficient complete downstream whole exome sequencing application.

The percentage of target coverage can be seen for the two kits tested. For the KAPA HyperPrep kit, coverage starts to drop off at high densities, becoming apparent at 10x, increasing at 20x, and dropping to <80 % at 50x. The Celero EZ DNA-Seq kit is able to consistently maintain greater than 98 % target coverage, which enables more accurate variant calling.

This figure shows a critical advantage of the Celero EZ DNA-Seq library preparation kit – the even coverage of target sequences. The KAPA HyperPrep kit offered insufficient coverage for large numbers of targets, while Celero libraries provided an even depth of coverage across all target sites




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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 Range

Input Range

10 ng - 500 ng

Multiplexing

Multiplexing

Up to 384-plex with Metaplex and Unique Dual Index adaptors

NuQuant compatible platforms

NuQuant compatible platforms

Qubit 2.0, 3.0 and 4. For plate reader compatibility and instructions contact Tech Support at techserv-gn@tecan.com.




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Tab 04 / Faq
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FAQ

Getting started

What materials are provided with Celero EZ DNA-Seq?

Celero EZ DNA-Seq includes all necessary buffers, primers and enzymes for library construction. SPRI purification beads and EvaGreen 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?

Celero 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 Celero EZ DNA-Seq with DNA from any organism?

Celero 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. We strongly recommend using high quality DNA with a A260:A280 ratio in excess of 1.8 and A260:A230 ratio in excess of 2.0. Use of DNA samples with lower ratios may result in low library yield.

General workflow

How much extra reagent is recommended when preparing the enzymatic fragmentation and adaptor ligation master mixes?

A minimum amount of overage should be used in master mixes to ensure the full nominal number of reactions in the kit. The amount of overage needed depends on sample batch size, pipetting accuracy, and viscosity of reagents. We have found that 12-15% extra volume in the enzymatic fragmentation and adaptor ligation master mixes is sufficient for most experiments.

Is it necessary to perform enzymatic fragmentation of my DNA?

Yes.

My input DNA samples are already fragmented (e.g. cfDNA, amplicons). Can I skip the enzymatic fragmentation step?

The enzymatic fragmentation step is required in the Celero EZ-DNA-Seq workflow. For sample types that are already fragmented, Celero DNA-Seq (Part No. 0360) is available with optional mechanical (Covaris) fragmentation and End Repair.

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

No.

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

My libraries have been stored at -20 °C. Can I still use NuQuant to quantify my libraries? Can I re-quantify libraries that have been stored at -20 °C?

Yes. Please contact Tecan NGS Technical Support for information on using NuQuant with previously frozen libraries.

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

We recommend using NuQuant to accurately quantify the final libraries for multiplex pooling. The final library pool concentration should be determined using a qPCR-based method before loading onto an Illumina sequencer. Please refer to section IV. F. in the User Guide for guidelines on library quantitative and qualitative assessments.

How many bases do Celero 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?

Celero 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?

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

Can Celero 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 Celero EZ DNA-Seq libraries?

The final Celero EZ libraries can be analyzed using standard pipelines. To remove adaptor sequences use standard TruSeq sequences for read trimming.




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Tab 05 / Literature
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