Next-generation sequencing (NGS) is a high-throughput technology that rapidly sequences large volumes of DNA or RNA, allowing for extensive genetic research in applications such as genomics, transcriptomics, and personalized medicine.
Image Credit: INTEGRA Biosciences
The usual NGS workflow can be separated into four main stages:
- Sample preparation
- Library preparation
- Quality control
- Sequencing
INTEGRA provides solutions to reduce the chance of human error, increase the precision and reliability of sequencing data, and deliver more efficient workflows at each stage of the process.
Step 1: Sample preparation: aliquoting and nucleic acid extraction
Aliquoting/reformatting
Primary tubes are used to collect a variety of samples, including blood and tissue. Aliquoting, or reformatting, is the process of transferring a portion of each sample from the primary tubes into a format appropriate for nucleic acid extraction or long-term storage.
INTEGRA’s solutions: INTEGRA recommends automating this process with the ASSIST PLUS pipetting robot and a VOYAGER adjustable tip spacing pipette.
The ASSIST PLUS accelerates science in the lab by completing routine pipetting activities hands-free. It is a versatile platform that can control all of INTEGRA’s VOYAGER and VIAFLO multichannel electronic pipettes, as well as the D-ONE single channel pipetting module.
If a user prefers a manual process, the VOYAGER without the ASSIST PLUS is available. Its adjustable tip spacing at the press of a button boosts productivity by allowing you to transfer several samples between different labware formats at once.
Nucleic acid extraction
Nucleic acids can be extracted using a variety of techniques, including magnetic beads. Following this, the extracted DNA and/or RNA is quality verified to ensure that it meets library preparation standards.
Fluorescence-based concentration assays, gel electrophoresis for integrity, and optical density measures to detect impurities (A260/A280 and A260/A230 ratios) are all examples of employed quality assessments.
INTEGRA’s solutions: If magnetic beads are being employed for nucleic acid extraction, INTEGRA proposes pairing MAG or HEATMAG modules with the ASSIST PLUS pipetting robot or VIAFLO 96 and VIAFLO 384 handheld electronic pipettes. This enables semi- or completely automated magnetic bead operations, which minimize manual errors while increasing productivity and reproducibility.
The MAG and HEATMAG modules have powerful magnets that automatically move up and down to collect beads quickly and precisely. In addition, HEATMAG provides integrated, regulated heating up to 65 °C, which can improve the efficiency of lysis and elution and maximize yields.
Step 2: Library preparation: preparing nucleic acids for sequencing
Library preparation is the process of converting extracted DNA or RNA fragments into a state suitable for sequencing. Accurate liquid handling is crucial for consistency and repeatability, especially when employing magnetic bead-based size selection to generate appropriate-sized DNA fragments.
NGS library preparation typically requires three basic steps:
- Fragmentation: DNA is fragmented using physical or enzymatic means, such as sonication or non-specific endonuclease, to create smaller, manageable bits for sequencing.
- NGS library generation: platform-specific adapters and multiplexing indexes (barcodes) are added during PCR.
- Size selection/clean-up: the PCR-generated library is purified to remove undesirable components such as primers, dimers, salts, and polymerases, resulting in a clean and sequencing-ready sample. Size selection can also be used to extract a certain DNA fragment size for sequencing.
Automated systems improve accuracy, reduce human error, and increase the precision and dependability of sequencing data while reducing procedures.
INTEGRA’s solutions: the MIRO CANVAS is a microfluidics technology that enables complete automation of NGS library preparation methods. It can complete enzymatic fragmentation, PCR processes, and bead clean-up in a single run.
To deploy magnetic bead operations, MAG or HEATMAG modules can be combined with INTEGRA’s benchtop liquid handling platforms, such as ASSIST PLUS, VIAFLO 96, or VIAFLO 384.
Step 3: Quality control: quantification, normalization, and pooling
Quality control ensures that the library is properly prepared, eliminating errors, increasing data accuracy, and enabling consistent sequencing results. This can be separated into three steps.
- Quantification: this involves measuring the concentration of DNA in the library to guarantee proper loading into the sequencer.
- Normalization: sample concentrations are adjusted to a uniform level to ensure equitable representation during sequencing.
- Pooling: this involves combining normalized samples in equal parts to achieve a balanced representation while increasing sequencing efficiency and throughput.
INTEGRA’s solutions: an ASSIST PLUS pipetting robot combined with a D-ONE module can automate the pipetting operations for quantification, normalization, and pooling. This system allows worry-free and hands-free processing of complex liquid handling tasks, with specialized software commands for easy worklist generation and pipetting step execution.
Step 4: Sequencing
NGS can be performed using a variety of technologies from companies such as Illumina, Oxford Nanopore, and PacBio. Read INTEGRA’s DNA sequencing methods articles to learn how the various approaches work, and then read INTEGRA’s Sanger sequencing vs NGS article to learn more about their benefits and drawbacks.
About INTEGRA Biosciences
INTEGRA provides innovative solutions for liquid handling and media preparation applications that serve the needs of its customers in research, diagnostics, and quality control laboratories.
Its instruments and plastic consumables are developed and manufactured in Zizers, Switzerland, and Hudson, NH, USA. To remain close to its customers, INTEGRA maintains a direct sales and support organization in several North American, European, and Asian countries, as well as a network of over 100 highly trained distribution partners worldwide.
In recent years, INTEGRA has focused on developing a new and technologically advanced range of lab devices that are simple to use and meet the ergonomic needs of its customers.
Today, the company is proud to offer the widest range of electronic pipettes on the market, covering everything from single channel pipettes to 384 channel benchtop instruments.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.net, which is to educate and inform site visitors interested in medical research, science, medical devices, and treatments.