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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.

List of available protocols for the Freedom EVO® NGS workstation

Related products:  TouchTools™  |  Freedom EVO®  |  Fluent®

Illumina qualified protocols

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

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Qualified

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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Verified

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 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.

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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.

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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.

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IonTorrent sequencing

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

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Co-Developed

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.

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There are so many things that you have to get right to create high quality libraries for reliable next gen 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 (1) eight pipetting channels to allow precise pipetting from 1,000 µl down to 0.5 µl. The workstation includes (2) three Inheco CPAC devices (to keep reagents cool and provide optimal conditions for the enzymatic steps), (3) an Inheco Thermoshake heated shaker, a (4) 96-position magnetic plate separator (Alpaqua 96S Super Magnet Plate) and (5) a RoMa Arm for efficient bead clean-up. In addition, the compact worktable provides storage space for (6) up to 12 tip boxes, 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 only 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.

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 correct initial conditions. A selection confirmation screen then provides a summary as a final check before the start of 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.

Fluent® Laboratory Automation Solution

Rapid technology advances have made NGS a cost-effective alternative to PCR or array-based techniques for many applications, allowing pooling of hundreds of samples in one sequencing run. The speed, accuracy and precision of Fluent make the system ideally suited to preparing NGS libraries from multiple 96- or 384-well plates.

Benefits include

  • Straightforward operation, allowing you to customize the user interface to show only the relevant options
  • Greater reproducibility between operators and over time, with high precision pipetting to reduce process variability
  • Flexibility to expand for increased throughput, allowing you to switch between 96- and 384-well formats even during a protocol
  • Extended walkaway operation thanks to easy integration of 1D/2D barcode readers, thermocyclers, microplate readers and a range of storage options for plates and tips

Fluent highlights

Intuition built right in

Built-in touchscreen interface guides users through daily tasks for fast, consistent operation. Integrated user management, including method permissions, ensures secure protocols and optimizes selection for each user.

Simply faster and more precise

Three independent, task specific arms move in parallel to simultaneously and efficiently automate methods. Sample preparation, labware movement to peripheral devices and plate-to-plate or reagent additions can all occur in parallel – maximizing performance.

Save reagents and samples

Save reagents and samples Adaptive Signal Technology™, a new generation of liquid level sensing, enables the use of smaller sample volumes, minimizes the risk of errors and provides exceptional automated error handling.

Integration options

Magnetic separation for bead-based NAP and PCR clean-up

Integrated DNA quantification and normalization

Heating and cooling for incubation steps

Vacuum separation for filter-based purification formats

Sample tracking and barcode reading

Thermocycling

Method Approval

Prevent unauthorized changes to validated protocols with the Method Approval function. Once any change is done to the protocol by an operator it cannot be executed any more until a key operator approves the changes.

Third-party device integration

FluentControl™ offers straightforward integration of third-party devices, with an ever-increasing library of drivers – including support of SiLA devices – and Zero-G one-touch teaching to simplify setup.

Enhanced productivity with Tecan consumables

Tecan offers a broad portfolio of high quality consumables – from disposable tips to troughs – in Tecan Standard, Tecan Pure and Tecan Sterile purity levels. Every product undergoes stringent testing to ensure reliable automation.

NGS sample preparation protocols require several quality control checks, such as quantitation of the amount of DNA available in a sample and review of the fragment size distribution in a sample.

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.

Fragment Analyzer INFINITY™ integration

The Fragment Analyzer INFINITY (FAI) from Advanced Analytical offers automated multichannel capillary electrophoresis, providing high resolution separation of 96 samples in parallel.

It can accurately size and quantify nucleic acid fragments and smears from 50bp to 50kb, and can hold two different gel reagents for seamless switching between applications and sample types.

The Infinite 200 PRO can be integrated on the workdeck, on a side extension or below the workdeck, ensuring an optimal fit into your genomics workflow.

Please see this technical note for further information on hardware and software integration.

Brochures

In conjunction with the ever increasing throughput of NGS instruments and the option to multiplex more and more samples, sample preparation has become a bottleneck. Automation of NGS sample preparation helps to reduce the workload, process more samples in parallel and increase the reproducibility of library preparations, while minimizing sample processing errors.

Fluent’s groundbreaking design delivers more capacity and increased speed for genomic workflows. This innovative solution offers excellent precision, outstanding throughput and increased walkaway times. Get more done in less time, with greater confidence.

Application Notes

High quality Ion AmpliSeq libraries for inherited disease and cancer research using custom panels of up to 96 samples in less than six hours.

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. It can accommodate 500 pg to 1 μg of input DNA, sheared by either mechanical or enzyme-­based methodsand prepare up to 96 libraries per run.

The automated method for TruSeq Nano DNA generates excellent quality data from as little as 200 ng – and up to 1 μg – of DNA. This allows samples with restricted DNA vailability, for example tumor biopsies, to be studied, helping to preserve precious sample material for future use. In comparison, the TruSeq DNA PCR-Free Library Preparation Kit, offers a time-saving alternative for more concentrated samples with initial quantities of between 1 and 2 μg of DNA, generating libraries with fewer coverage gaps by the elimination of PCR-induced bias, providing greater access to the genome.

Illumina’s 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.

HLA gene sequencing is a technique of growing interest, as it allows improved determination of the HLA profile of tissue donors and acceptors compared to classic techniques, such as serotypical analysis.

Illumina’s TruSeq technology delivers stranded information, identifying which DNA strands a given RNA transcript has derived from. This information ensures increased confidence in transcript annotation and raises the percentage of alignable reads, ultimately reducing the sequencing cost per sample. This application note describes automation of the TruSeq RNA library preparation kits.

The Illumina Nextera Rapid Capture Exome and Expanded Exome kits provide a fast and simple, all-in-one sample preparation and hybridization-based enrichment process for identifying coding variants in targeted resequencing studies.

Agilent's SureSelectXT 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.

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.

User Guides

Prepare Ion AmpliSeq Libraries using the Tecan Freedom EVO® NGS Workstation

User guide for NEBNext Ultra II DNA Library Prep Kit

User guide for Illumina TruSeq Nano DNA and TruSeq DNA PCR-Free library preparation

User guide for Nextera XT DNA library

User guide for TruSight HLA v2 Sequencing Panel library preparation

User guide for Illumina TruSeq Stranded mRNA library preparation

User guide for Illumina TruSeq Stranded Total RNA library preparation

User guide for Illumina TruSeq RNA Access library preparation

User guide for the Illumina Nextera Rapid Capture library preparation

User Guide for Agilent SureSelect XT Target Enrichment System

User guide for NimbleGen SeqCap EZ Library SR with KAPA™ HTP Library Preparation