What is targeted Next Generation Sequencing?
What is targeted region sequencing?
Targeted NGS is a widely used NGS approach to provide high accuracy genomic data for human, animal, plant and microbial research. Through the use of targeted gene sequencing panels and specifically designed primers which target the regions of interest (ROIs), the process of library generation and enrichment, make it possible to interrogate key areas of the genome. In this way, specific mutations in a given sample are studied.
Often it is not necessary to sequence the entire genome when in fact you have specific ROIs. By focusing only on ROIs, sequencing resources are not wasted on areas that you are not interested in. It also allows multiple samples to be interrogated at the same time, thereby reducing associated sample sequencing cost.
As this targeted NGS approach provides a more cost effective way of sequencing clinically relevant regions of the genome, it is often used in healthcare and clinical settings to identify genomic aberrations associated with different health conditions. It enables greater depth of coverage and therefore improved sensitivity of ultra-rare variants including single nucleotide polymorphisms (SNPs), insertions and deletions (indels) and copy number variants (CNVs), compared to whole genome sequencing (WGS) or whole exome sequencing (WES). See Figure 1 for more examples of what targeted NGS can be used for.
The deep sequencing enabled by the targeted NGS approach is a key advantage of this technique, whilst also providing a rapid turnaround time from sample collection through to bioinformatic NGS reporting. The data analysis stage is also simplified due to sequencing efforts being focused only on target regions of the genome (figure 2).
Figure 1. What can targeted NGS be used for? Figure 2. What are the benefits of targeted NGS?
What are the steps of a targeted NGS assay?
The workflow of a targeted NGS assay involves DNA extraction, library preparation, high-throughput sequencing and bioinformatic data analysis (Figure 3). This process is highly similar to other NGS techniques but targeted NGS involves an extra step of target enrichment. The process of target enrichment can be via hybridization capture or amplicon sequencing – find out the differences between the two in Table 1.
Figure 3. Flow diagram outlining the steps of the targeted NGS process.
1. Nucleic acid extraction
Targeted NGS studies can be done using nucleic acid samples from any biological sample. In a clinical setting, it is often genomic DNA (gDNA) from fresh frozen (FF) or formalin fixed paraffin embedded (FFPE) tissue, and/or cell-free DNA (cfDNA) from blood, urine, sweat or cerebral spinal fluid liquid biopsies.
For nucleic acid extraction:
- The DNA, RNA or cfDNA is isolated from the biological sample using an extraction kit. The kit used at this stage should enable the highest quality and quantity of input material for the NGS workflow.
- Quality controls are also necessary ahead of the next step of library preparation, to ensure the assay proceeds with a viable concentration and purity of material.
2. Library preparation
In this step of library preparation, the nucleic acid sample is prepared for sequencing through the creation of sequencing libraries. It is during this stage that target enrichment can be done via hybridization capture or amplicon sequencing (Table 1).
For hybridization capture, fragmentation of the DNA sample may be required to ensure all DNA fragments entering the sequencer are similar in size, for example, when using gDNA. The fragmentation step is avoided when carrying out a NGS assay using cfDNA as these fragments are already short in size at ~167 bp.
- Sequencing adapters are ligated to the ends of the DNA fragments, which enables attachment to a flow cell. If multiple samples are being sequenced in the same run, the adapters are likely to also contain a unique molecular identifier (UMI) sequence, which enables individual libraries to be identified during multiplexing, which is often used to save both time and cost.
- Biotinylated oligonucleotide probes are designed to bind to the target regions and streptavidin-beads are used to pull down the probes and associated targets. Targets are captured using a magnet and off-target sequences are washed away, prior to library amplification using PCR.
In amplicon sequencing, primers are designed based on the ROIs and multiplex PCR is used to amplify the target sequences. After multiplex PCR, background cleaning is performed as well as indexing PCR to prepare the libraries for sequencing.
Table 1. Comparison between hybridization capture and amplicon sequencing.
|Features||Hybridization Capture||Amplicon Sequencing||Lowest amount of input material||1 ng||10 ng|
|Number of processing steps||higher||lower|
|Number of targets||Unlimited - based on panel size||Limited - based on the number of amplicons|
|Time to results||slower||faster|
|Cost per sample||higher||lower|
|Sensitivity||< 1% VAF||5% VAF|
3. Next Generation Sequencing
There are different sequencing approaches including pyrosequencing and sequencing by ligation, but it is sequencing by synthesis (SBS) that is most commonly used. Illumina reports that ~90% of sequencing studies use SBS.
- In SBS, the libraries are loaded onto a flow cell, which is coated with millions of oligonucleotide sequences that are complementary to the sequencing adaptors, this then goes into the sequencer for massive parallel sequencing.
- The DNA sequence is read one nucleotide at a time. Chemically modified bases synthesize a complementary strand to each fragment of DNA, the fragments are amplified to create clonal clusters.
- After the incorporation of each fluorescently tagged nucleotide, the flow cell is imaged, and it is the emission wavelength and intensity from each clonal cluster that is recorded.
- For paired-end sequencing, the initial forward DNA strands are read, and then the process is repeated for the reverse strand. The number of reads produced is dependent on the type of sequencer used.
4. Data analysis
After NGS, bioinformatic pipelines are often required to make sense of the sequencing data.
- Reads are assembled based on size and agreement of the paired ends, and base calling is used to identify the exact sequence of bases and predicts the likelihood of that base being called correctly.
- The sequences are aligned to a reference genome build. By configuring the data in this way, it is possible to detect genomic variants associated with different health conditions or diseases.
Why is targeted NGS used for liquid biopsy samples?
The benefits of targeted NGS make it the ideal approach for analyzing patient DNA samples; often these samples are limited in their abundance. In the case of liquid biopsies, it is often cell-free DNA samples including cell-free circulating tumor DNA (ctDNA) released from cancer cells or fetal cell-free DNA (cffDNA) from maternal blood samples that are required for evaluation. Being able to accurately detect and measure these samples is critical to being able to inform patients of health conditions and provide the correct treatment pathway as well as monitor their responses and recovery.
Targeted NGS can be used to confidently call mutations down to 0.1% variant allele frequency (VAF) from as little as a few nanograms of cfDNA. To be able to deliver this level of sensitivity, very deep sequencing is required, often raw read depths of around 20,000x. For this depth of coverage, sequencing efficiency is critical, the designed probes within the NGS assay must deliver high on-target rates and uniformity of coverage, ensuring only ROIs are sequenced and therefore not wasting precious sequencing resources.
Would you like to carry out a targeted NGS assay?
If you would like to carry out a targeted NGS assay, it is firstly important to identify your ROIs. If your ROIs are genomic regions associated with common health conditions then you could choose to use an NGS panel with pre-existing content. Nonacus panels have been designed by NGS expects to achieve uniform coverage and high on-target rates. To explore the oncology NGS panels offered by Nonacus click here, for constitutional genetics panels click here and for prenatal healthcare panels click here.
Alternatively, you may want to make modifications to an existing panel or design a new panel containing all of your own targets. This can easily be done through the Nonacus cloud-based Panel Design Tool. The Panel Design Tool will provide you with instant feedback on your panel design. Once your NGS panel has been chosen, you will be able to use it within the Cell3™ Target hybridization and capture technology workflow developed by Nonacus.
From sample collection through to bioinformatic analysis, Nonacus provide an optimized NGS workflow to ensure sensitive detection of genomic variants from both gDNA and cfDNA samples; with a streamlined workflow and high-performance probes, your targeted NGS assay can be efficient, effective and affordable.