Targeted sequencing: the definition, application and methods


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Targeted sequencing: the definition, application and methods

What is targeted sequencing?

Targeted sequencing is a genomic analysis method that uses specialized probes to acquire a specific DNA sequence of interest to a customer and then perform high-throughput sequencing

Targeted sequencing achieves the goal of saving time and cost-effectiveness by only using the genomic region of interest. Compared with WGS (whole-genome sequencing) (for DNA), targeted sequencing yields a smaller database and therefore requires easier data analysis, but this method may miss previously unknown mutations.


How to obtain the specified target DNA sequence?

Here we introduce two main methods:   

One is the capture method. A series of oligonucleotide probes are designed for the specific genomic region of interest, and the enrichment of specific genomic regions is achieved by hybridization of probes with whole genome DNA fragments.

The second is the PCR amplification method. Primers are designed for the region of interest, and then the DNA sequence of the region to be tested is amplified by PCR.

Targeted Resequencing Applications

Here we introduce two clinical applications of targeted sequencing:

The first is to establish a suitable target therapy. In a sequencing experiment to detect mutations in lung cancer, Feng and others used targeted sequencing to identify mutations in 737 gene loci from 45 lung cancer patients, and found that mutation often occurred in egfr, kras, pik3ck and TP53 genes, and in some samples, there are two or three mutations in these key genes. Different types of lung cancer correspond to different gene mutations, and targeted sequencing can identify these mutations and can be used to guide individualized tumor sequencing and targeted therapy. In Non-small cell lung cancer, anti-EGFR targeting therapy often involves a single point mutation, but in malignant gliomas, anti-EGFR targeting therapy is regulated by complex epigenetic genetics.

The second application is that targeted sequencing has opened a new direction in disease diagnosis of unknown etiology, especially in prenatal diagnosis. Traditional prenatal screening is the use of amniocentesis, has a great risk of miscarriage. Fetal chromosomal abnormalities can now be detected by noninvasive prenatal screening (NIPS), i.e. by detecting whether the maternal serum contains free DNA (Cell-free DNA, Cfdna) associated with fetal chromosomal abnormalities. The sequencing of NIPS genes in pregnant women showed that the results of gene sequencing were consistent with those of conventional nuclear chromosome analysis.

Prenatal testing of Down syndrome and Edward's syndrome in high-risk pregnant women showed that the false positive rate of large scale parallel sequencing for cell free DNA sequencing was significantly lower than that of traditional antenatal screening (0.3% vs 0.6%, Down syndrome; 0.2% vs 0.6%, Edward's syndrome), Positive predictive rates also had significant differences (45.5% vs 4.2%, Down syndrome;40.0% vs 8.3%, Edward's syndrome), DNA sequencing results are significantly better than traditional prenatal screening, and targeted sequencing only need to extract the blood of the mother to check, which is convenient and quick.

Targeted sequencing can obtain genetic information from specified target areas, greatly improving the efficiency of research on these target areas, and significantly reducing the cost of research. It is mainly used to identify and study structural variations in coding regions associated with disease and population evolution. Target area sequencing can be used to search for complex diseases such as cancer, diabetes, obesity and other pathogenic genes and susceptible genes, in order to overcome these difficult diseases of mankind to provide a new way.

Targeted sequencing is also often used in these areas, such as, Cancer Gene Sequencing, Condition Screening, Genetic Disease Research, NGS for Drug Development, Forensic Genomics and 16S rRNA Sequencing. Sequencing the bacterial 16S ribosomal RNA gene is a common amplicon sequencing method used to identify and compare bacteria present within a given sample. It is useful for studying complex microbiomes or environmental samples.

Key Targeted Resequencing Method---Targeted Gene Panels

Targeted panels contain predefined probe sets focused on specific genes of interest. Gene panels are available with preselected content or can be custom designed according to regions of interest.

Targeted gene panels are suitable for the analysis of specific mutations in a given sample. These collections, which focus on certain functions, contain a set of genes or gene areas be associated with known or to be predicted disease phenotypes

The new generation sequencing (NGS) provides the scalability, speed, and resolution required to meet the target gene for the assessment. Multiple genes in many samples can be evaluated at the same time so that multiple independent analyses can be run to save time and reduce costs. In addition, targeted sequencing produces less data and is easier to manage than other methods that have wider range, such as whole-genome sequencing, making it easier to analyze.

The collection of finished genes contains an important gene or gene region associated with a disease or phenotype, selected from literature and expert advice. These collections focus on genes with the greatest potential relevance, save resources and minimize data analysis considerations. The finished gene combination can be used to study a variety of diseases, such as cancer, hereditary diseases, heart disease and autism.

Technical process:

In general, a complete target sequencing is divided into the following steps:

1.Sample preparation: Sample Selection, Genomic DNA extraction

2.Sequencing Library Preparation: Target area DNA capture or expansion, construct sequencing library

3.Sequencing: On-machine sequencing, data quality control, data filtration or quality evaluation

4.Genome contrast splicing: Splicing of reference genomes

5.Annotation analysis: Localization and functional annotation of target region sequence on genome

6.Health Letter Analysis: SNP, inDel, Identification and analysis of synonymous or non-synonymous mutant genes, advanced information analysis of complex diseases

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