Quick overview: Next Generation Sequencing

Next Generation Sequencing (NGS) is a revolutionizing laboratory method that can sequence an entire human genome by determining the order of the chemical building blocks in a DNA molecule.

NGS methods have opened the door to a whole new world for scientists and is game-changing in e.g., life science and health tech. It can be used for everything from studying microbiomes, identifying bacteria, studying infectious disease, discovering novel pathogens, and cancer research to investigating genetic diseases.

As opposed to Sanger sequencing, the lab work can be carried out in just one day with equipment that will fit into most labs which makes it much more accessible. In addition, NGS can sequence millions of fragments simultaneously thus allowing for a fast high throughput of samples and making sequencing more cost-effective.

The NGS Workflow

NGS covers a wide range of methods within Genomics, Epigenetics, Transcriptomics, Proteomics, Liquid Biopsy NGS and much more.

In general, the basic NGS workflow involves:

• Library preparation
• Sequencing
• Data analysis

Step 1: Library Preparation

Library preparation prepares DNA and RNA samples to be compatible with an NGS sequencing platform by creating a so-called sequencing library.

Sequencing libraries are typically created by fragmenting DNA into several pieces, adding adapter sequences and indexes to both the 3’ and 5’ end of the DNA strands, amplifying the DNA fragments with PCR and purifying the DNA.

The adapter sequences enable attachment of the DNA fragments to the flow cell during sequencing while the indexes make it possible to assign the sequenced DNA fragments to the correct samples during data analysis.

Step 2: Sequencing

During sequencing, the libraries are loaded onto a flow cell and placed on the sequencer.

The DNA fragments binds to the surface of the flow cell and are then amplified , creating clusters of millions of copies of single-stranded DNA.

The single strands that are not attached to the flow cell’s oligonucleotides gets washed away and the remaining DNA strands are then sequenced.

Step 3: Data Analysis

After sequencing, the sequence reads are assigned to the appropriate samples based on the indexes added. They are then aligned to a reference genome. Further analysis such as single-nucleotide polymorphism, phylogenetics or metagenomic analysis is now possible using a bioinformatics pipeline.

Liquid handling in the NGS Workflow

An NGS workflow involves several pipetting tasks, including, but not limited to:

1. Pipetting various reagents and buffers
2. Transferring the sample DNA
3. Transferring master mixes
4. Sample normalization
5. Adding index adapters to samples
6. Bead-based purification with multiple washing steps

Depending on the protocol, it can amount to over 300 pipetting tasks per 96 well plate when using an 8-channel pipette and around 2500 when using a single-channel pipette. This is an extremely heavy workload to carry out manually.

Consequently, laboratories of all shapes and sizes choose to implement pipetting robots. Robots can be a huge advantage for preventing cross-contamination, increasing throughput, avoiding errors, and ensuring a healthy work environment without work-related injuries.

We contribute to this with the pipetting robot, flowbot® ONE, that can easily make the NGS assays flexible, user-friendly, and cost-effective while protecting laboratory staff from being overburdened.