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Celero™ DNA-Seq library preparation workflow

Simple DNA-Seq library preparation kit enables library construction in three steps. NuQuant library quantification technology included providing accurate library molarity in seconds.




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Tab 01 / Overview
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DNA-Seq libraries in 3 simple steps

The Celero DNA-Seq library preparation kit is an innovative system designed to help researchers streamline library preparation for Illumina sequencers. Celero features a fast, easy-to-use, addition-only workflow that eliminates post-ligation bead purification. Also included is Tecan's breakthrough library quantitation method, NuQuant®, that saves both time and cost in measuring library concentration for pooling. With the proven DimerFree® technology you can use from 10 ng to 500ng of intact DNA with a single kit without the worry of GC bias.

Celero DNA-Seq library preparation kit offers several unique benefits:

  • Short, simple workflow:  NGS library preparation in 3 steps with reduced hands-on time, no adaptor or template dilution or post-ligation purification.
  • No adaptor dimers: get more informative reads compared without multiple bead clean-ups.
  • Flexible multiplexing: dedicated dual index (up to 384) or Unique Dual Index adaptors (up to 192) for each reaction included with the kit.
  • Integrated library quantification: quickly determine library molarity without the need for costly and time-consuming processes.
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  • Automatable: enabled on multiple liquid-handling platforms

NuQuant app for Qubit® can be downloaded here on Github.

Applications

  • Whole genome sequencing
  • Exome sequencing
  • Microbiome
  • Amplicon sequencing




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Tab 02 / Highlights
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A simplified addition-only workflow

  • Celero is an addition-only workflow with 3 master mixes and only 1 bead purification clean-up following library construction
  • Elimination of post-ligation bead purification saves time compared to other methods
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Celero DNA-Seq has a shorter workflow compared competitor workflows

Celero DNA-Seq has the simplest and shortest workflow. Master mixes are provided at each step and bead purifications are eliminated after ligation step for an easy, streamlined library preparation.

High quality libraries for DNA-Seq

  • Streamlined workflow enables generation of high quality libraries
  • Reduced base bias
  • Provides better sequencing efficiency of high and low GC organisms
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High quality data with minimum PCR bias across a broad range of GC content

Celero provides superior sequencing efficiency of high and low GC organisms. gDNA from E. coli (51 % GC), R. sphaeroides (69 % GC) and S. aureus (33 % GC) were mixed to equal mass and sequenced with Celero DNA-Seq and kits from Companies K and I. The percentage of each genome is given as a fraction of total reads. PCR amplified Celero libraries demonstrate the same bacterial balance as the PCR-free control. Company K has a significant bias against the A/T-rich genome

Innovative DNA fragmentation method reduces base bias

Celero DNA-Seq provides reduced base bias. 10 ng of an equal mass mixture of Covaris sheared gDNA from E. coli (51 % GC), R. sphaeroides (69 % GC) and S. aureus (33 % GC) was sequenced with Celero DNA-Seq and kits from Companies K and I. Graphs from FastQC showing the base distribution (first 25 bases shown in graph; A - Green, T - Red, C - Blue, G - Black) demonstrate that Company K has a significant footprint in the first 10 bases and a high A/T bias. Company I also has a noticeable footprint in the first 8 bases.

Lower index hopping than other methods

  • Significantly lower index hopping observed (0.24%) compared to other current methods (e.g. expected range 0.5-2.0%) using a patterned flow cell
  • Unique Dual Indexes available for identifying index hopping events
  • Multiplexing up to 384 samples available with Metaplex barcodes

NuQuant library quantification: Measuring molarity in minutes

  • Obtain molar concentrations of individual libraries immediately for multiplex pooling
  • A single standard makes reference measurements simple
  • More reproducible than qPCR-based library quantitation methods for sample pooling
  • NuQuant library quantification app freely available for Qubit® 2.0, 3.0 and 4
Show more

NuQuant is more accurate than traditional methods

NuQuant has the lowest variability for library quantitation. A set of 8 libraries was distributed to 6 different users at multiple institutions. Users were asked to use NuQuant and their own preferred method of library quantitation (number denoted by n). For each library, and each quantification method, the percent coefficient of variation (%CV) of molar concentration was calculated. NuQuant produced the lowest variation, followed by qPCR. Bioanalyzer gave the highest variation.

NuQuant determined library molarity

Strong correlation between NuQuant molarity and read numbers. Two users prepared 8 libraries each from 10 ng of genomic DNA sheared using a Covaris and amplified either 10 or 13 cycles to produce both lower and higher library concentrations. All measurements were performed in duplicate, across two users, for a total of 16 libraries. Purified libraries were quantified by NuQuant, then equal volumes of all libraries were pooled and sequenced on one lane of an Illumina sequencer.




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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 ng - 1 μg




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Tab 04 / Faq
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FAQ

What materials are provided with Celero DNA-Seq?

Celero 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 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.

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 DNA-Seq with DNA from any organism?

Celero 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?

We strongly 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. This kit is designed for use with DNA samples of high molecular weight with little or no evidence of degradation. FFPE DNA may be compatible with the PCR workflow. When using degraded samples, we recommend using inputs of 200 ng or greater. While it is impossible to guarantee satisfactory results with all degraded samples, this system may work with many samples that are moderately degraded.

Is it necessary to fragment my DNA prior to End Repair and Adaptor Ligation?

Yes.

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

No.

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.

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

Please refer to section V. H. for guidelines on quantitative and qualitative assessment.

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

The adaptors add 136 bp to the library.

What sequencers are compatible with your libraries?

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

Can Celero 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.

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 DNA-Seq libraries?

The final Celero 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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Resources

Type
Title
No
User Guide
M01477 V2
Product Sheet
400987 V1.1
Application Note
401056 V1.1
Application Note
401102 V1.1
Product Sheet
400985 V1.1
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For research use only. Not for use in diagnostic procedures.