Targeted sequencing — providing adequate depth of coverage for areas of interest through enriching the target DNA or RNA molecules or the depleting of molecules that are unwanted — is a useful method to generate a deeper and broader understanding of coverage for insightful and cost-effective analysis.
Targeted sequencing provides unprecedented insight into specific sections of the genome. It is an extremely versatile application that may be used to investigate a wide number of illness areas, including oncology, hereditary diseases, immunology, and infectious diseases. This tool enables precise and efficient targeting of individual genes, coding areas, and even parts of chromosomes. Clinical laboratory testing has included this technology, which generates test results for producing precision medicine.
One of the challenges plaguing the genomics community is the continuing accumulation of vast volumes of raw sequencing data that has yet to be completely and efficiently translated and interpreted in order to aid in the advancement of research, diagnosis, and treatment of diseases on a larger scale. Even with today’s lower sequencing prices, a whole genome sequencing approach is practicable only in a few circumstances, like basic research, detection of rare diseases, and population genetics. A focused or targeted method would be ideal for outlining guiding therapy selection and disease progression in the clinical setting or screening DNA samples in large quantities in industrial applications. Additionally, sequencing a full genome or exome can be too costly in terms of its laboratory operations plus bioinformatics infrastructure for data storage and processing. As such, targeted sequencing has now become essential for the advancement of research and precision medicine.
Compared to whole genome sequencing or WGS, targeted sequencing is less expensive. Additionally, it offers a more in-depth examination of results than WGS or other survey methods. Furthermore, it enables deeper sequencing, and the increased depth of coverage aids in the avoidance of incorrect interpretations of the sequencing of data. This means that by restricting the flow cell’s sequencing real estate for only regions which is of interest, the genomic sequences could be enriched by up to a millionfold when compared to DNA sequencing. This is critical for certain applications in which the typical coverage of 10x or 100x obtained through DNA sequencing or even large exome sequencing may not always be sufficient. Similarly, greater confidence in these low-frequency variant calls can be obtained with deep sequencing via NGS rather than Sanger sequencing.
Target sequencing, due to sensitivity, offers a significant edge in variant calling for cancer research, disease-associated mutation identification, single-gene disorders, and gene expression studies. Additionally, targeted sequencing of specific regions enables the detection of uncommon diseases’ causative genes. Targeted sequencing’s specialized methodology allows its implementation in targeted therapy and customized medicine endeavors. Targeted resequencing of the polymorphic human leukocyte antigen (HLA) gene, for example, aids in HLA typing, which is necessary for hematopoietic stem cell or solid organ transplantation matching.
What are the Advantages of Targeted Sequencing?
Targeted sequencing is best suited for:
- Concentrating on specific regions of interest
- Analyzing microbial genes in a targeted manner
- Designed to detect rare variants, sequence-focused substance from a large number of samples in parallel via multiplexing
- Attaining comprehensive coverage while yielding a smaller, more workable amount of data
- identifies causative variants such as SNPs and InDels throughout multiple genomic regions
The Workflow of Targeted Sequencing
Methods and technology for targeted sequencing
By isolating and sequencing target sections of the genome from a sample pool, targeted resequencing is indeed a high-throughput NGS approach that is increasingly being utilized to find causative mutations among target content in populations.
There are various ways for targeted sequencing, each of which is well-suited for a particular purpose. Amplicon sequencing and hybridization capture are the most frequently used methods.
The primary distinction of the techniques being used is the procedure used to enrich the samples. Additionally, the stage at which samples would be multiplexed is a differentiating factor. Multiplexing, alternatively referred to as pooling, enables the processing of several samples concurrently, thereby saving both money and time. Multiplexing entails the addition of a barcode or index to samples to facilitate their identification following sequencing.
Enrichment of the Target
With great specificity and sensitivity, sequencing enrichment will target genomic regions of interest, enabling the discovery of both common and rare variants.
Exome sequencing is perhaps the most frequently used approach for targeted resequencing. Exome sequencing enriches just the protein-coding or exon sections of the human genome, which are thought to contain the majority of phenotype-causing variations. Exome sequencing enables complete coverage of coding areas.
Gene-panel Sequencing
Panel sequencing is a method for sequencing several genomic regions of interest using next-generation sequencing technology. Pre-designed or custom-designed panels can be used to pinpoint cancer genes or DNA areas associated with inherited disorders or certain phenotypes. Gene panels significantly minimize sequencing costs, time, and analytical effort by sequencing only the genes of interest.
Amplicon Sequencing
PCR can be used to amplify smaller target DNA areas. Ultra-deep sequencing of the PCR amplicons generated is a focused technique that is typically utilized for variant screening and analysis of somatic mutations in tumor samples.
Multiplexing options enable the analysis of multiple genes from many samples in parallel at a cost and time equivalent to a single-gene experiment. This significantly simplifies the finding of variants in populations and enables the investigation of entire genes along with their surrounding regions and co-variations.
PCR Simplification for Sanger Sequencing
Enrichment and capture of targets utilizing next-generation sequencing technologies with high-coverage sequencing is a frequently used technique for targeted sequencing. Alternatively, PCR amplification can be employed in conjunction with Sanger sequencing to sequence a small number of targeted areas. This approach discovers base locations between different genes, SNPs, and minor deletions and insertions in genomic DNA sequences. This is a frequently used technique to identify heterozygotes, identify genomic rearrangements, and detect unusual variations.
16S rRNA Sequencing and ITS Sequencing
Sequencing the hypervariable sections of the 16S ribosomal RNA gene or the ITS region is a closely related amplicon sequencing method used to detect and classify bacteria or fungus present in a given sample. Pooling PCR amplicons helps to simplify the study of complicated microbiomes or environmental materials. Full-length 16S rRNA gene amplicon sequencing enables precise phylogenetic characterization and quantification of microbes down to the genus or even species level.
Personalized Sequencing
Prior to sequencing, the regions of interest are enriched, which simplifies subsequent data interpretation. This approach can be used with any organism for which a reference genome is available.