Step 1: Sample preparation: Aliquoting and nucleic acid extraction

Aliquoting/re-formatting

Various sample types, such as blood or tissue, are collected in primary tubes. Aliquoting or reformatting involves transferring a portion of each sample from the primary tubes into a format suitable for nucleic acid extraction or long-term storage.

Our solutions: We recommend using the ASSIST PLUS pipetting robot to automate a VOYAGER adjustable tip spacing pipette for this step. The ASSIST PLUS accelerates science in your lab by performing routine pipetting tasks hands free. It's a versatile platform that can automate all of our VOYAGER and VIAFLO multichannel electronic pipettes, as well as the D-ONE single channel pipetting module.

Read our application note with ready-to-use scripts to learn more about how the ASSIST PLUS can be used to reformat samples from tubes to plates.

If you prefer a manual workflow, the VOYAGER can also be used without the ASSIST PLUS. Its adjustable tip spacing at the press of a button increases productivity by allowing you to move multiple samples between different labware formats simultaneously.

Nucleic acid extraction

Nucleic acids can be extracted using various methods, including magnetic beads. After this, the extracted DNA and/or RNA is first quality checked to meet library preparation requirements. Quality assessments may include fluorescence-based concentration measurements, gel electrophoresis for integrity, and optical density measurements to detect contaminants (A260/A280 and A260/A230 ratios).

Our solutions: If you're working with magnetic beads for nucleic acid extraction, we recommend using MAG or HEATMAG modules in combination with the ASSIST PLUS pipetting robot or VIAFLO 96 and VIAFLO 384 handheld electronic pipettes. This enables semi- or fully automated magnetic bead workflows that eliminate manual errors and optimize productivity and reproducibility. The MAG and HEATMAG modules feature strong magnets that automatically move up and down for fast and precise bead collection. In addition, HEATMAG offers integrated, controlled heating up to 65 °C, which can increase the efficiency of lysis and elution to maximize yields.

Step 2: Library preparation: Preparing nucleic acids for sequencing

Library preparation converts the extracted DNA or RNA fragments into a format suitable for sequencing. Accurate liquid handling is critical for consistency and reproducibility, particularly when using magnetic bead-based size selection to obtain DNA fragments of the correct size.

NGS library preparation commonly involves 3 main steps:

– Fragmentation: The DNA is fragmented using physical (e.g. sonication) or enzymatic (e.g. non-specific endonuclease) methods to generate smaller, manageable pieces suitable for sequencing.
– NGS library generation: Adapters specific to the NGS platform are added, and indexes (barcodes) for multiplexing may be incorporated during PCR.
– Size selection/clean-up: The PCR-generated library is purified to remove unwanted components – such as primers, primer dimers, salts and polymerases – ensuring a clean, sequencing-ready sample. Size selection can also be used to isolate the desired DNA fragment size for sequencing. 

Automated systems enhance accuracy, minimize human errors and improve the precision and reliability of sequencing data, while streamlining workflows.

Our solutions: The MIRO CANVAS is a microfluidics platform that enables full automation of NGS library prep protocols. It can perform enzymatic fragmentation, PCR steps and bead clean-up all in one run.

Read our application notes on how the MIRO CANVAS can be used to automate NGS library prep protocols.

Alternatively, you can combine MAG or HEATMAG modules with our ASSIST PLUS, VIAFLO 96 or VIAFLO 384 benchtop liquid handling platforms to implement magnetic bead workflows.

Read our application note with ready-to-use scripts on how the ASSIST PLUS can be used to automate NGS size selection workflows.

Step 3: Quality control: Quantification, normalization and pooling

Quality control ensures that the library is prepared correctly, reducing errors, improving data accuracy and enabling reliable sequencing outcomes. This can be divided into 3 steps:

– Quantification: Measurement of the DNA concentration in the library to ensure that the correct amount of material is loaded into the sequencer.
– Normalization: Adjustment of sample concentrations to a uniform level, ensuring equal representation of each sample during sequencing.
– Pooling: Combining normalized samples in equal parts to ensure balanced representation while maximizing sequencing efficiency and throughput.

Our solution: The pipetting steps of the quantification, normalization and pooling can be automated using an ASSIST PLUS pipetting robot in combination with a D-ONE module. This solution offers worry- and hands-free processing of complex liquid handling tasks, with dedicated software commands for easy worklist generation and implementation of pipetting steps.

Read our application note with ready-to-use scripts about how the ASSIST PLUS can be used to automate DNA normalization workflows.

Step 4: Sequencing

NGS can be carried out with a variety of different technologies, from companies such as Illumina, Oxford Nanopore and PacBio. Read our blog on DNA sequencing methods to learn how the different techniques work, and take a look at our Sanger sequencing vs NGS blog to learn more about their pros and cons.