Celero DNA-Seq Library Prep - Tecan Genomics (2024)

Innovative chemistry, ultimate flexibility

Celero DNA-Seq Enz and Celero DNA-Seq Mech are innovative systems designed to help researchers streamline DNA-Seq library preparation when starting with either intact or pre-fragmented DNA. Based on proprietary chemistry from NuGEN®, these kits include DimerFree technology, which eliminates the need to titrate adaptor concentration, and NuQuant technology, a rapid way to quantify your DNA-Seq libraries.

Additionally, the kits offer higher reagent volumes for complete automation readiness. Multiple qualified methods are available for Tecan's DreamPrep® NGS and NGS Compact, using Celero DNA-Seq library preparation kits which allow users to streamline their automation efforts with a single point of contact. The reagents can also be used with other automation platforms.

The newly updated Celero kits now offer 3 workflows with the same kits, giving the user ultimate flexibility for their project and goals.

A workflow for every project

Celero product line consists of the following kits, listed here in the Tecan e-shop.

These kits are needed for every workflow with the option for built-in quantification.

Celero DNA-Seq Library Prep Kit Components

Core Module: Celero DNA-Seq Enz and Celero DNA-Seq Mech

The core module for enzymatic workflow contains the reagents for the enzymatic fragmentation and ligation. Alternatively, the core module for mechanical workflow contains reagents to be used with sheared DNA, for end repair and ligation.

NuQuant Module

The NuQuant module contains the necessary PCR enzyme mix and primers for library amplification and integration of NuQuant.

Adaptor Plates: 10-nt UDI adaptor plates for upto 384-plex multiplexing

The adaptor plates provide the unique dual index adaptors for multiplexing samples for sequencing.

Using the complete Celero kit combination for enzymatic or mechanical fragmentation; users can choose to run any of the 3 workflow options shown below.

Celero DNA-Seq Library Prep - Tecan Genomics (1)

Celero DNA-Seq Enz and Celero DNA-Seq Mech library preparation kits are designed to work with both PCR (Single or dual bead purification) and PCR free workflows.

PCR workflow with dual bead purification: Ideal for projects needing built-in quantification and automation.
With built-in NuQuant quantification, the dual bead purification workflow achieves complete library preparation including quantification in 3 hours. Further, readily available automation script on DreamPrep or other instruments achieves 96-192 samples throughput in 3-3.5 hrs! (Sample Input 10 - 500 ng)

PCR workflow with single bead purification: Ideal for manual workflow processes and quicker turnaround time.
If you have your own quantification system and are preparing libraries manually, use this workflow to save time and effort by reducing purification steps. (Sample Input 10 - 500 ng)

PCR-Free workflow: Start with 200-500 ng of DNA and use the PCR-Free workflow for in-depth investigation of your sample.

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

Highlights include

  • Simple and Flexible Workflows: PCR and PCR-Free with Single or Dual Bead Purification
  • No adaptor dimers: Proprietary DimerFree technology included in all kits efficiently removes unwanted adaptor dimers without requiring any additional hands-on time.
  • High-throughput multiplexing: Offers up to 384, 10-nt UDI adaptors for efficient multiplexing
  • Time-saving: No adaptor or template dilution and no post-ligation purification step with single-bead purification workflow leading to reduced hands-on time
  • Broad Sample Types and Inputs: Start with human gDNA, ds cDNA, FFPE DNA, or amplicons with 10-500 ng input (PCR) and 200-500 ng (PCR-Free)
  • Integrated library quantification: By adding NuQuant to your workflow, determine library molarity in 6 minutes, eliminating the need for separate quant methods
  • Automatable: Celero kits provide automation-friendly reagent volume with higher overages, optimized for DreamPrep NGS (Fluent 780 workstation). Additional pre-optimized scripts for other platforms are also available.

Applications

  • Whole-genome sequencing
  • Exome sequencing
  • Targeted enrichment
  • Microbiome

Reliable performance regardless of workflow

The flexibility of the Celero DNA-Seq kits allows users to start with either intact or pre-fragemented DNA. Additional flexibility is provided through the ability to select an ideal protocol for the laboratory (PCR vs. PCR-free, manual vs. automation, etc.). Here we show that the Celero DNA-Seq kit provides high quality and similar data regardless of the workflow used.

We mixed equivalent amounts of E. coli (51% GC), R. sphaeroides (69% GC), and S. aureus (33% GC) genomic DNA to generate a control sample that could be used across all the Celero DNA-Seq workflow options to evaluate performance and GC bias.

The bacterial control sample was used as input into the Celero DNA-Seq Enz module. The same control sample was fragmented to 300 bp using a Covaris S2 and quantified before using as input into the Celero DNA-Seq Mech workflow. The Celero DNA-Seq workflows were tested with either 10 ng or 100 ng inputs for the PCR workflow or with 200 ng input for the PCR-free workflow. Overall, the data shows consistent performance across all three workflows with minimal adaptor artifact and >95% alignment, >98% uniformity, and <2% chimera. Additionally, we also see even read coverage across a broad GC range as shown in the GC Bias graph.

Workflow

Module

Input

# PCR cycles

% adaptor artifact

PCR workflow with
Dual bead-purifications
(includes NuQuant)

Celero Mech

10 ng

10

0.0

100 ng

7

0.0

Celero Enz

10 ng

9

0.2

100 ng

6

0.2

PCR workflow with
Single bead-purification

Celero Mech

10 ng

10

0.0

100 ng

7

0.3
Celero Enz

10 ng

9

0.1

100 ng

6

0.1

PCR-free workflow

Celero Mech

200 ng

N/A

0.5

Celero Enz

200 ng

N/A

0.2

High library quality across the different Celero DNA-Seq workflows. In-house blended DNA from E. coli (51% GC), R. sphaeroides (69% GC), and S. aureus (33% GC) was used for performance testing of different workflows with both the Celero DNA-Seq Mech and Enz modules. All three workflows, provided high quality libraries with minimal adaptor artifacts (library between 50-150 bp).

Workflow

Module

Input

% alignment

% duplicates

% chimera

PCR workflow with
Dual bead-purifications
(includes NuQuant)

Celero Mech

10 ng

98.60.20.3

100 ng

98.50.31.1

Celero Enz

10 ng

97.60.30.6

100 ng

97.70.41.5

PCR workflow with
Single bead-purification

Celero Mech

10 ng

98.60.20.2

100 ng

98.60.20.8
Celero Enz

10 ng

97.60.30.4

100 ng

98.20.41.1

PCR-free workflow

Celero Mech

200 ng

98.30.20.7
Celero Enz

200 ng

98.20.31.6

Sequencing metrics for the different Celero DNA-Seq workflows. All the different Celero DNA-Seq workflows tested provided high quality sequencing data with high alignment rates and low duplicate and chimera rates.

Average Uniformity for Celero Mech Kits

Celero DNA-Seq Library Prep - Tecan Genomics (2)

Average Uniformity for Celero Enz Kits

Celero DNA-Seq Library Prep - Tecan Genomics (3)

Both bead clean-up workflows provide consistent sequencing efficiency of high and low GC organisms. 100 ng Covaris-sheared gDNA produced by equivalent blend of 3-bacterial mix from E. coli (51 % GC), R. sphaeroides (69 % GC) and S. aureus (33 % GC) was used for library prep with A) Celero DNA-Seq Mech and B) Celero DNA-Seq Enz modules. Libraries were sequenced on a MiniSeq™ system (Illumina) using 2 x 75 bp PE reads and data was downsampled to equivalent sequencing depth for each library. All three bacterial genomes showed high average uniformity regardless of workflow. The E. coli uniformity is slightly lower (~0.5%) due to its larger genome. The uniformity and alignment for the PCR workflows is similar to the data genetrated with the PCR-free protocol (data not shown) indicating minimal PCR introduced bias.

GC Bias

Celero DNA-Seq Library Prep - Tecan Genomics (4)

Even read coverage across a broad GC range. 10 ng (left) and 100 ng (right) of gDNA from E. coli (51% GC), R. sphaeroides (69% GC) or S. aureus (33% GC) were sequenced from Celero DNA-Seq Enz (pink and blue) and Celero DNA-Seq Mech (orange and green) with single (pink and orange) or dual (blue and green) bead cleanup, respectively. Result for the PCR-free protocol are similar (data not shown).

NuQuant Library Quantification Method: Measuring Molarity in minutes

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 Dual bead purification.

Features of NuQuant method include:

  • Accurately measure molar library concentration without the need for library size analysis (e.g. Bioanlyzer)
  • Quantify only sequence able 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
  • 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

Celero DNA-Seq Library Prep - Tecan Genomics (5)

NuQuant is more accurate than traditional methods

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

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.

Zoom

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.

Zoom

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.

Zoom

NuQuant is more accurate than traditional methods

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

Zoom

NuQuant saves time and money compared to traditional quantification methods

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

Zoom

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.

Zoom

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.

Zoom

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

Product Specifications

Specification

Specification/Description

Workflows

Workflows

  • Enzymatic or mechanical fragmentation
  • PCR and PCR-Free
  • Single bead OR dual bead purification

Adaptors

Adaptors

Up to 384, 10-nt UDI adaptors for high throughput multiplexing

Applications

Applications

  • Whole genome sequencing
  • Exome sequencing
  • Target enrichment
  • Microbiome analysis

Sample Input

Sample Input

10 ng – 500 ng (PCR workflow)
200 ng – 500 ng (PCR-Free workflow)

Sample Types

Sample Types

Human: gDNA, ds cDNA, FFPE DNA, amplicon
Non-Human: any source DNA

Sequencing

Sequencing

Compatible for all Illumina® Platforms

Automation Specifications

Specification

Specification/Description

Automation

Automation

  • Automation friendly reagent volume with low dead volumes
  • Optimized for DreamPrep® NGS (Fluent® 780 workstation)
  • Additional pre-optimized scripts for other platforms available

Sample Throughput

Sample Throughput

96-192 per day as standard on DreamPrep (T-GN automation team available to develophigher scale)

Automated Install Time

Automated Install Time

3 days on all liquid handling robots (including validation with reagents + control samples)

Sample Input

Sample Input

10 ng – 500 ng (automated applications tested down to 1 ng)

Hands on time

Hands on time

20 minutes

Liquid Handling Time

Liquid Handling Time

1.5 hrs

Thermal Cycling Time

Thermal Cycling Time

1.35 hrs

Total Run Time

Total Run Time

3-3.5 hours

FAQ

Getting started

What materials are provided with Celero Enz and Mech Kits?

Celero kits includes all necessary buffers, primers and enzymes for library construction. SPRI purification beads, low TE buffer 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.

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 DNA-Seq with DNA from any organism?

Celero DNA-Seq (Enz and Mech) 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.

General workflow

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

No.

General workflow (For Celero DNA-Seq Enz)

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 DNA-Seq Enz Workflow. For sample types that are already fragmented, Celero DNA-Seq Mech Workflow (Part No. 30188861) is available with optional mechanical (Covaris) fragmentation and End Repair.

General workflow (For Celero DNA-Seq Mech)

Is it necessary to fragment my DNA prior to End Repair and 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 toevaluate the product?

Electrophoretic methods such as Agilent Bioanalyzer or Fragment Analyzer instruments are useful for interpreting library size,but are not recommended for library quantification. Please refer to Section IV. G. Quantitative and Qualitative Assessment of the Library of the User Guide for more information.

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

10 nt UDI adaptors add 140 bp to the library.

Sequencing recommendations

What sequencers are compatible with your libraries?

Celero DNA-Seq libraries are compatible with Illumina sequencing platforms.

What is the maximum allowable mismatch that can be used during sample demultiplexing?

Maximum allowable mismatch during demultiplexing is 1 when utilizing 10 nt UDI plates (UDI-A/B/C/D).

What kind of error correction is used to minimize the impact of sequencing errors during index sequencing reads?

For additional details on the index sequence 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.

Resources

Type

Title

No

User Guide

Celero DNA-Seq Enz Library Preparation Protocol

M01580V1.0

User Guide

Celero DNA-Seq Mech Library Preparation Protocol

M01579V1.0

Product Sheet

Celero DNA-Seq Library Preparation

402923V1.0

Application Note

NuQuant

401102V1.1

Product Sheet

NuQuant Library Quantification

400985V1.1

Poster

Poster Fluorescence-based Method for accurate molar quantification of NGS libraries in minutes

Safety Data Sheet

10nt UDI Adaptor Plate SDS

10ntUDIMSDSV1.0

Safety Data Sheet

Celero DNA-Seq Core Module SDS

CeleroMSDSV1.0

Safety Data Sheet

NuQuant NGS Library Quantification Module SDS

NuQuantMSDSV1.0

Celero DNA-Seq Library Prep - Tecan Genomics (2024)

FAQs

How much DNA is needed for library prep? ›

For human DNA samples and other large complex genomes, the recommended DNA input is between 100-500ng. For small genomes, the DNA input amount can be reduced to as low as 1 ng. The number of PCR cycles should be modified in accordance with the protocol.

How long does DNA library prep take? ›

Manual library preparation takes between 4 and 8 hours of work. Typically, a full day is required for preparation of libraries for DNA and two days for mRNA sequencing libraries.

How to prepare a DNA library? ›

Typically, a conventional DNA library construction protocol consists of 4 steps:
  1. Fragmentation of DNA.
  2. End repair of fragmented DNA.
  3. Ligation of adapter sequences (not for single-molecule sequencing applications)
  4. Optional library amplification.

How much genomic DNA for sequencing? ›

The recommended input for library construction is 100–200 ng of genomic DNA, which should be delivered in a volume of 15–50 ul. Lower quantities of DNA can also be used with this kit.

What is the minimum library concentration for sequencing? ›

Sequencing Library Requirements

The standard requirements for library submission are at least 15 ul volume at a concentration of 5 nM (e.g., 2.3 ng/ul given a 700 bp library).

What is the minimum amount of DNA required for sequencing? ›

If you are interested in genome or metagenome sequencing on any of the Illumina sequencers such as the Illumina MiSeq or Illumina NovaSeq, the recommended amount of DNA is 50 ng-500 ng. If the genome you are trying to sequence is large or complex, we strongly recommend submitting at least 100 ng of good quality gDNA.

How long does it take to get whole genome sequencing results? ›

How Long Does Whole Genome Sequencing Take? Because of the technical complexity and multi-step nature of whole genome sequencing, the standard process typically can take from 10 to 12 weeks once the lab has recieved your sample. If the lab is especially busy, processing time may extend up to 16 weeks.

What is the first step in library preparation for whole genome sequencing? ›

DNA fragmentation strategies. The first step in NGS library preparation for Illumina systems is fragmentation of DNA into the desired size range, typically 300–600 bp depending on the application.

What is DNA library prep for NGS? ›

The library preparation process involves converting a genomic DNA sample (or cDNA sample) into a library of fragments which can then be sequenced on an NGS instrument.

How is library preparation done? ›

Overview. Library preparation is the first step of next generation sequencing. It allows DNA or cDNA to adhere to the sequencing flow cell and allows the sample to be identified. Two common methods of library preparation are ligation-based library prep and tagmentation-based library prep.

What technique is used to prepare genomic library? ›

The Genomic Library or gene bank is constructed by a shotgun experiment where the entire genome of the cell is cloned in the form of random and unidentifiable clones. It uses the chain termination method (Sanger's sequencing) to sequence the DNA molecules.

How much does it cost to get your entire genome sequenced? ›

There are many providers that offer whole genome sequencing tests in the United States; many of them offer prices that range from $999 to as low as $399.

Can I get my entire genome sequenced? ›

However, as sequencing costs and data storage requirements have continued to drop, whole genome sequencing (WGS) has become more feasible, though it still is not a widely offered service. WGS translates all of the 3 billion DNA base pairs that make up an entire human genome into a file made up of letters.

What is a good DNA concentration for sequencing? ›

Thus, the optimal concentration for sequencing is 1.4 ng/µl. Since we request template in 10 µl, you would submit 14 ng purified PCR product.

What is the minimum amount of DNA needed for DNA analysis? ›

To be valid the DNA profile must have at least 10 loci obtained.

How much DNA is needed for sufficient data? ›

DNA Extraction and Analysis

To perform a forensic DNA analysis, DNA is first extracted from a sample. Just one nanogram of DNA is usually a sufficient quantity to provide good data.

How much DNA is needed for a sample? ›

Dry DNA samples

If you are working with human DNA (genome size ~3000 Mbp) or a species with a genome size in the range of 2000 – 3500 Mbp, we require 10 ng of good quality DNA per sample per SNP (except for DNA quantified using PicoGreen, where a minimum of 5 ng per sample per SNP is acceptable).

How much DNA should I send for sequencing? ›

How Much Sample Should I Send? For DNA sequencing, the amount is dependent upon the type of the sample you have. Soil and tissue samples require at least 0.25g. We recommend sending a few grams extra especially if multiple extraction replicates per sample are requested.

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