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NGS sample preparation

Automated library preparation, quality control and pooling

From just a few samples a week to hundreds a day, automation of NGS sample preparation allows you to reproducibly create high quality libraries for reliable next generation sequencing.

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Tab 01 / DreamPrep™ NGS Solution
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DreamPrep™ NGS

Walkaway automation solution for NGS library prep

DreamPrep NGS is a fully optimized solution for walkaway NGS library prep and QC. It minimizes manual intervention, saving time and delivering consistent, sequencing-ready libraries for a wide range of research applications.

Watch the free webinar!

DreamPrep NGS

 

DreamPrep NGS combines the Fluent® Automation Workstation, Infinite® F Nano+ plate reader and Tecan library preparation kits to provide sequencing-ready libraries with unprecedented speed and accuracy. This groundbreaking approach offers preparation, quantification, normalization and pooling of NGS libraries in one automated workflow.

Tecan’s innovative NuQuant® library quantification method uses the integrated Infinite reader to enable fully automated QC without sample loss in under six minutes, eliminating the need for slow and costly qPCR- or capillary electrophoresis-based QC.

Each DreamPrep NGS workstation is supplied with example methods* for Tecan reagent kits, including AnyDeplete™ mediated transcript depletion of unwanted reads from sequencing data.

System highlights

  • DNA-Seq libraries, normalized and ready to sequence, in less than four hours
  • mRNA-Seq libraries, quantified and ready to sequence in nine hours instead of 14
  • Integrated, full library QC in less than six minutes without sample loss
  • Addition-only workflows for DNA-Seq, plus minimal intervention for RNA-Seq
  • Single point of contact for automation, reagents, consumables, application support, etc.
  • Intuitive, touchscreen-based user interface that can be customized to your needs

 

* Example methods have not been validated. Please make a copy of these methods to customize them to accommodate and validate your workflow according to your desired intended use and laboratory protocols. Tecan makes no claims regarding the performance of the example methods.

 

Rapid advances in technology have made NGS a reliable and simple option for genomic assays, but library preparation remains a challenge for high throughput applications. The speed, accuracy and precision of the Fluent platform makes it ideally suited to preparing NGS libraries, reducing process variability and ensuring greater reproducibility between operators and over time. The system’s three independent, task-specific arms operate in parallel to provide fast, efficient processing. Its built-in touchscreen interface guides users through daily tasks and can be easily customized to suit your laboratory and workflow needs for straightforward operation. Together with integrated user management and Method Approval functions, this ensures secure protocols and optimal performance.

On-deck thrmocycling for longer walkaway runs

Integrated plate reader for QC without sample loss

Magnetic separation for bead clean-ups

Intuition built right in

Simply faster and more precise

Save reagents and samples

Prevent unauthorized changes to validated protocols with the Method Approval function.

Enhanced productivity with Tecan consumables




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Tab 02 / Reagents
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Tecan NGS library prep reagents for Illumina sequencing

Tecan has developed and optimized its own NGS library preparation reagents for a broad range of sample types. These reagents have been verified on Tecan automation platforms to give you a complete solution, from samples to readily normalized libraries.

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Universal Plus™ mRNA-Seq

The Universal Plus mRNA-Seq library preparation kit features a robust end-to-end workflow with a wide input range for analysis of coding transcripts. Universal Plus mRNA libraries can be prepared, quantified, normalized and pooled in less than nine hours on the Fluent® Automation Workstation, compared to more than 14 hours for manual or automated library preparation and qPCR quantification with other kits.

Key features of this mRNA-Seq library preparation kit include:

  • Broad dynamic input range from 10 ng to 1 µg
  • DimerFree® technology eliminates adaptor dimers
  • NuQuant® library quantification method integrated at no additional cost to accurately quantify library concentrations (in nM)
  • Integrated AnyDeplete® technology aids the removal of unwanted transcripts post library preparation, to maximize informative sequencing reads
  • Available with up to 192 unique dual index (UDI) adaptors for increased multiplexing
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Trio RNA-Seq™

Trio RNA-Seq offers a complete streamlined library preparation solution that combines three core technologies to capture rare transcripts and low abundance pathogens. Trio’s proven amplification technology (single primer isothermal amplification, SPIA®) generates more molecules for library preparation compared to standard RNA-Seq workflows, preserving precious biological information from limited samples.

Key features of this RNA-Seq library preparation kit include:

  • RNA sequencing solution ideal for liquid biopsy, FFPE, nasal swabs, and other challenging samples with low or degraded inputs
  • Broad dynamic input range from 500 pg to 50 ng
  • DimerFree® technology eliminates adaptor dimers
  • Maximize informative sequencing reads by removing unwanted transcripts post library preparation with AnyDeplete®
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Celero™ EZ DNA-Seq

The Celero EZ DNA-Seq library preparation kit is a simple, single tube, addition-only workflow with just one post-amplification bead purification step. Celero DNA libraries can be prepared, quantified, normalized and pooled in less than four hours on the Fluent® Automation Workstation.

Key highlights of this DNA-Seq library preparation kit include:

  • Broad dynamic input range from 10 to 500 ng
  • Addition only, three-step workflow
  • Robust optimization-free enzymatic fragmentation
  • DimerFree® technology eliminates adaptor dimers
  • NuQuant®* library quantification method integrated at no additional cost, to accurately quantify library concentrations (in nM)
  • Available with 384 UDI adaptors for increased multiplexing

*NuQuant technology available with Plate A (92 UDI indexes) only. 

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Ovation® Ultralow System V2

The Ovation Ultralow System V2 is a DNA-Seq library preparation kit for ultra-low inputs. This kit includes our proprietary DimerFree® technology, which eliminates excess adaptor dimers to get more informative reads from your sequencing data. Ultralow V2 DNA libraries can be prepared in less than three hours on the Fluent® Automation Workstation.

Key features include:

  • DNA sequencing solution ideal for cell lines including fresh or FFPE tissue, liquid biopsy, cell-free RNA and ultra-low input RNA samples
  • Ultra-low input range from 10 pg to 100 ng
  • DimerFree technology eliminates adaptor dimers
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Allegro® Targeted Genotyping V2

Allegro Targeted Genotyping provides a fast, scalable, cost-effective approach for targeted sequencing of a wide variety of organisms using next generation sequencing. Allegro uses the patented Single Primer Enrichment Technology (SPET) method to specifically target regions of interest, providing information-rich sequencing data by capturing a SNP- or genotype-specific data point for every on-target sequencing read. The result is unparalleled sequencing efficiency, leading to rapid scalability and the lowest available cost per data point.

Key features

  • Input range from 10 to 100 ng
  • Simple, single-tube assay that can be completed in less than 24 hours
  • Hundreds of barcodes and increased multiplexing enable cost-saving interrogation of SNPs
  • Ability to design panels for any sequenced genome
  • High throughput multiplexing options with 3072 Metaplex® barcodes
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Illumina qualified protocols

Tecan has collaborated with key research centers and Illumina to develop automated NGS sample preparation protocols that have been optimized, tested and sequencing verified, to ensure excellent performance in any laboratory.

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TruSeq Nano DNA

The TruSeq Nano DNA Sample Preparation Kit enables efficient investigation of samples where the amount of DNA available is limited. The automated workflow for this kit on the Freedom EVO® NGS workstation includes simple bead-based size selection, and can be completed in less than a day with minimal hands-on time for up to 48 samples.

Download Application Note

TruSeq PCR Free

The TruSeq DNA PCR-Free Sample Preparation Kit enables time-saving investigation of DNA samples. The user-friendly TouchTools™ interface guides the operator through automation set-up to deliver highly reproducible, sequencing-ready DNA libraries for single read, paired-end and indexed sequencing with minimal user intervention.

Download Application Note

Nextera XT

The Nextera XT DNA Library Preparation Kit enables the effective construction of high quality libraries from PCR amplicons, plasmids and small genomes, using a protocol optimized for DNA inputs from as little as 1 ng.

Download Application Note

TruSeq Stranded mRNA

RNA sequencing (RNA-Seq) is a powerful method for discovering, profiling and quantifying RNA transcripts. Using Illumina next generation sequencing technology, stranded information identifies the DNA strand from which a specific RNA transcript was derived, providing increased confidence in transcript annotation and visibility into antisense regulation.

Download Application Note

TruSeq Stranded Total RNA

Automation of the Illumina TruSeq Stranded Total RNA protocol on the Freedom EVO NGS workstation enables the conversion of total RNA into libraries of template molecules of known strand origin with minimal user intervention. The Freedom EVO NGS workstation delivers high quality libraries, with minimal risk of cross-contamination.

Download Application Note

TruSeq RNA Access

The TruSeq RNA Access library preparation protocol is the ideal solution for cost-effective sequencing of formalin-fixed paraffin-embedded (FFPE) archival tissue samples. The integrated enrichment steps can accommodate low starting concentrations of DNA, and the automated workflow allows the construction of up to 48 libraries in parallel, generating high quality libraries with limited user intervention and minimal risk of cross-contamination.

Download Application Note

Nextera Rapid Capture

The automation-friendly workflow of the Nextera Rapid Capture protocol combined with the Freedom EVO NGS workstation provides a faster, more efficient solution for library preparation and exome enrichment. Using the automated Nextera Rapid Capture Exome protocol, sample preparation can be completed with minimal hands-on time in just a day and a half, generating sequencing data results the following day.

Download Application Note

TruSight® HLA v2.0 Sequencing Panel

Interest in HLA gene sequencing is growing, as it offers better determination of the HLA profile of tissue donors and recipients compared to classic techniques, such as serotypical analysis.

The Illumina TruSight HLA v2.0 Sequencing Panel is a reliable and simple assay designed to generate NGS libraries for HLA profiling. Automation of the workflow on the Freedom EVO NGS workstation allows sequencing of 11 HLA loci for up to 24 samples, minimizing the risk of cross-contamination and human errors during library preparation.

Download Application Note

TruSeq ® Custom Amplicon Low Input

Amplicon sequencing is a targeted method for identification and analysis of variations in specific regions of the genome. Sequencing of PCR products offers cost-effective variant identification and characterization. Automation of the TruSeq Custom Amplicon Low Input library preparation workflow on the Freedom EVO NGS workstation provides a fast and efficient solution. This setup enables generation of high quality libraries from low input samples – starting with as little as 10 ng of genomic DNA.

Verified for Illumina sequencing

Tecan has developed, tested and sequencing-verified NGS sample preparation protocols in collaboration with NGS sample preparation kit vendors, to give you time to focus on analyzing sequencing results.

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NEBNext Ultra II Directional RNA

The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina uses the ‘dUTP` method to produce excellent yields and high quality directional libraries even from low quality RNA. Up to 96 libraries can be prepared at a time from a broad range of input material, 10 ng - 1 µg total RNA for poly(A) mRNA enrichment, 5 ng - 1 µg for rRNA depletion.

Download Application Note

NEBNext Ultra II DNA

The NEBNext Ultra II DNA library prep workflow combines the end repair and dA-tailing steps with minimal clean-¬up to reduce sample preparation time. Up to 96 libraries can be prepared per run, and multiplexed using, for example, the NEBNext Multiplex Oligos for Illumina.

This protocol allows highly reproducible library preparation from a wide range of input DNA concentrations from 500 pg to 1 μg, as well as FFPE samples, offering flexible processing of 1 to 96 samples with minimal user intervention.

Download Application Note

SureSelect XT Target Enrichment

Agilent’s SureSelect XT Target Enrichment System provides a complete portfolio of exome kits for humans and other species, as well as custom kits for both DNA and RNA capture.

Click here for an application note describing an automated protocol for parallel processing of up to 48 samples using the SureSelect XT Target Enrichment System on the Freedom EVO NGS workstation.

Download Application Note

NimbleGen SeqCap EZ Library

Roche NimbleGen’s SeqCap EZ Library is a solution-based capture system enabling enrichment of the whole exome or custom regions of interest from DNA sequencing libraries. The SeqCap EZ protocol covers exome designs, gene panels and custom content designs, according to Roche NimbleGen specifications.

Click here for an application note describing an automated protocol for NimbleGen SeqCap EZ with KAPA™ HTP Library Preparation in combination with NimbleGen SeqCap Adapter Kits A and/or B. The protocol allows preparation of up to 96 libraries, and capture of up to 24 library pools.

Download Application Note

IonTorrent sequencing

Sequencing-verified, flexible protocols for Ion AmpliSeq™ library preparation, developed in collaboration with Thermo Fisher Scientific.

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Ion AmpliSeq libraries and panels

The Ion AmpliSeq Library Kit 2.0 enables rapid NGS library preparation for 12-25,000-plex PCR in a single well.

Automated library preparation of up to 96 samples can be performed on the Freedom EVO NGS workstation in less than six hours using 10 ng of DNA or RNA. The TouchTools operator interface minimizes hands-on time and training requirements, guiding users through every stage of protocol selection and worktable set-up.

Download Application Note




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Tab 03 / Qualified NGS protocols
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    NGS_Dreamprep-FreedomEvo_300x300

 

NGS_Dreamprep-Fluent_300x300

 

Supplier Kit Freedom EVO® NGS workstation DreamPrep™ NGS
Tecan
DNA-Seq Celero™ DNA-Seq    
  Celero™ EZ DNA-Seq    
  Ovation® Ultralow System V2    
RNA-Seq Universal Plus™ mRNA-Seq    
  Universal Plus Total RNA-Seq    
  Revelo™ RNA-Seq    
  Trio RNA-Seq™    
  Ovation SoLo RNA-Seq    
Targeted genotyping Allegro® Targeted Genotyping V2    
 
Illumina
DNA-Seq Illumina DNA Prep    
  Nextera XT    
  TruSeq® Nano DNA    
  TruSeq DNA PCR-Free    
RNA-Seq COVIDSeq Test   Illumina qualification in progress
  TruSeq® RNA access (now TruSeq® RNA exome)    
  TruSeq Stranded mRNA    
  TruSeq Stranded Total RNA *NEW*   Water tested
Forensics ForenSeq® DNA Signature Prep    
Targeted enrichment 16S Metagenomic Sequencing    
  Nextera DNA Exome    
  TruSight® Cancer    
  TruSight Cardio    
  TruSight Inherited Disease Sequencing Panel    
  TruSight Rapid Capture    
HLA typing TruSight HLA v2 Sequencing Panel    
 
NEB
DNA-Seq NEBNext® Ultra™ II DNA    
RNA-Seq NEBNext Ultra II Directional RNA Poly(A) mRNA Isolation    
  NEBNext Ultra II Directional RNA rRNA depletion    
 
QIAGEN
DNA-Seq QIAseq® FX DNA    
RNA-Seq QIAseq Targeted RNA Panels    
Targeted enrichment QIAseq Targeted DNA Panels    
 
Roche
DNA-Seq KAPA® HyperPlus    
  KAPA HyperPrep    
  KAPA HTP Library Prep    
RNA-Seq KAPA RNA HyperPrep with RiboErase (HMR)    
  RNA HyperPrep with mRNA Capture    
 
Agilent
  SureSelect® QXT Target Enrichment    
  SureSelectXT Target Enrichment    
  Haloplex® HS Target Enrichment System    
 
Bioo Scientific
RNA-Seq NEXTflex® Small RNA-Seq Kit v3    
 
Thermo Fisher Scientific
Targeted enrichment Ion AmpliSeq® Library 2.0     
  Oncomine™ Comprehensive Assay v3M    
 
GenDx
HLA typing NGSgo® HLA typing     
 
Quantabio
DNA-seq sparQ® DNA library kit   Water tested
 
Twist
Targeted enrichment Human Core Exome   Water tested
 

 Method qualified/verified by vendor1
 Method qualified/verified by Tecan1
 Method custom developed2

1) Automated method developed according to the kit vendor’s published protocol. Libraries prepared with these methods meet the kit vendor’s specifications and/or perform comparably to those prepared manually. Sequenced libraries were assessed either by the vendor (vendor qualified/verified) or by Tecan (Tecan qualified/verified).
2) Automated method developed according to specific customer need. Libraries tested by the customer.




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Tab 04 / Freedom® Evo NGS workstation
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There are so many things that you have to get right to create high quality libraries for reliable next generation sequencing, so why have unnecessary complexity in your liquid handling?

Automation of NGS sample preparation on the Freedom EVO NGS workstation increases throughput and reproducibility, guiding the user through each step of protocol selection and worktable set-up with the TouchTools™ operator interface.

Freedom EVO NGS workstation

The Freedom EVO NGS workstation is equipped with innovative air displacement pipetting technology, using eight pipetting channels (1) to allow precise pipetting from 1,000 µl down to 0.5 µl. The workstation includes three Inheco CPAC devices (2) to keep reagents cool and provide optimal conditions for the enzymatic steps, an Inheco Thermoshake heated shaker (3), a 96-position Alpaqua 96S Super Magnet Plate magnetic plate separator (4) and a RoMa Arm (5) for efficient bead clean-up. In addition, the compact worktable provides storage space for up to 12 tip boxes (6), allowing longer unattended runs.

Precision: The Freedom EVO’s precision automation ensures the reproducibility necessary for high quality results, with a range of qualified sequencing-verified protocols available.
Flexibility: Don’t be constrained by batching requirements. Whether you need to process 6, 13, 19, 40 or even 96 samples, only run what you need, saving on reagents.
Simplicity: You don’t need to be an automation expert. With the simple touchscreen interface, you can get started right away, set up your runs quickly and be confident in your workflows.

NGS Sample preparation Freedom EVO with Touchtools

TouchTools operator interface

The TouchTools application starter screen lets the operator select the desired protocol with the touch of a button. Step-by-step user instructions help to avoid potential errors during worktable set-up to ensure the correct initial conditions. A selection confirmation screen then provides a summary as a final check before starting the run, making it easy for anyone in the lab to use the instrument.

Installation and support

The Freedom EVO NGS workstation is supplied with one of the standard protocols available at the time of installation, including water testing and user training. Additional protocols can be installed at any time.

Tecan Journal Articles

The Leiden University Medical Center has integrated the Freedom EVO® NGS workstation into its molecular diagnostic testing and clinical research workflows. Taking advantage of this preconfigured solution, the center was quickly able to commission the platform for routine testing, while still having the flexibility to develop new protocols and conduct research studies.

The University of North Carolina (UNC) has automated its major sample preparation protocols for next generation sequencing on a Freedom EVO® workstation, enabling more efficient processing.

The GeT genomics facility near Toulouse has been developing a series of automated NGS protocols in collaboration with Tecan – including Illumina’s TruSeq® DNA and RNA kits, as well as Bioo Scientific’s NEXTflex™ PCR-Free Modules – on two Freedom EVO® workstations, helping to improve throughput and reliability for a wide range of projects.




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Tab 05 / Detection
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Sensitive and fast quantification

The Infinite® F Nano+ multimode reader offers absorbance and high sensitivity fluorescence reading modes in one compact instrument. It combines the high sensitivity of fluorescence assays and the speed provided by reading plate formats of up to 384 wells.

 

Read more

NGS library quantification is a crucial step for a successful sequencing run. Accurately determining the number of sequenceable molecules is highly significant for new high capacity sequencers, such as the NovaSeq system.

Tecan NuQuant technology

Tecan NuQuant technology

Tecan NuQuant® is a novel library quantification method that accurately measures molar library concentration without the need for separate fragment size analysis. This proprietary method uses a fluorescent label that is incorporated into the library molecules during library preparation. NuQuant is independent of library size, allowing library molarity to be directly measured using fluorometers, such as the Infinite F Nano+ multimode reader.

Integrated library QC in less than six minutes

NuQuant library quantification on the Infinite F Nano+ plate reader eliminates the need for additional manual qPCR or capillary electrophoresis steps, saving significant time, resources and valuable library material.

After final purification of the DNA libraries in a 96-well plate on the Fluent ® Automation Workstation, the fluorescence of the libraries and standards is measured on the Infinite plate reader. The molarity determined for each library can then be used for balanced pooling and subsequent sequencing on Illumina instruments.

Other quantification assays

Other quantification assays

Reader integration for quantification

DNA quantification can be achieved by measuring an aliquot with a fluorescent dye, such as PicoGreen® or QuantiFluor™, or by measuring the absorbance of the entire DNA sample. DNA quantification and normalization workflows typically include the following steps

  • Set-up of quantification assay
  • Determination of the DNA concentration in a reader
  • Calculation of a dilution scheme to achieve the target concentration for each well
  • Dilution of the DNA samples to the desired concentration.
Read more




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Tab 06 / Literature
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