Targeted Capture and Sequencing: An Accurate and Efficient Method for Human Pathogen Detection
Next-Generation Sequencing or NGS has provided scientists alternative approaches in understanding DNA. The technology has come a long way from the simple sequencing technique developed by Sanger in 1977 and continuous improvements are being done to better cater to the needs of researchers. One of the recent advances to NGS is Target-and-Capture Sequencing or Capture-Seq. Capture-Seq allows the retrieval of large genomic fragments that are needed for downstream analysis such as for phylogenetic studies. This method has been mostly used to capture exons in the genome of an organism in order to identify genetic variants and even novel members of a particular gene family. This technology is said to be more advantageous than long-read sequencing because it increases the detectability of isoforms and lessens the ambiguity of the results. It is also used to sequence genes with very low copy numbers.
Capture sequencing applies the use of transcript enrichment techniques wherein DNA or RNA probes are hybridized to the target region in the isolated DNA and then it is followed by high-throughput sequencing. It can be directly applied after nucleic acid isolation and library preparation. Short, single-stranded DNA or RNA oligonucleotide probes specific to the target regions are added to the mix and allowed to bind to the DNA sample after this, the unbound fragments are washed out from the sample. After amplification, NGS is performed to determine the sequence of the target genes and bioinformatic analysis follows to identify the sequences. Capture sequencing is also commonly used in pathogen detection and classification.
Target-and-capture sequencing was first designed for human genomic studies to sequence the human exome. Eventually, it was used to sequence human infectious pathogens such as parasites, fungi, bacteria, and viruses. Some human parasites are said to have low sequence proportions compared to their human host and with the help of capture sequencing, the enrichment of parasite sequences has been successfully used, giving way to increased read accuracy and parasite detection. Similarly, fungal pathogens are also difficult to detect in human host samples due to low sequence proportions, and with Capture-seq enrichment, the analysis of fungal genes increased in reads and genome coverage. On the other hand, for bacteria and viruses, enrichment of sequences is essential in order to isolate and clearly identify the target pathogen, particularly in clinical samples.
The figure below summarizes the application of Capture-Seq in pathogen detection, identification, characterization, and determination of virulence/resistance determinants (source: Gaudin and Desnues, 2018). For pathogen detection, this technology is able to detect even low-copy number pathogens and even detect different pathogens at the same time. Also using the information gathered after this technique, strain typing can be done to establish phylogenetic relationships and variations. It can also be used to detect genomic rearrangements and the presence of mobile genetic elements. Lastly, virulence and antibiotic resistance genes can be directly identified using the technology.
Capture sequencing provides an efficient way to detect, identify, and characterize human pathogens especially the ones that are particularly difficult to isolate and sequence. Targeted enrichment also ensures that only the specific gene of interest will be selected and undergo high-throughput sequencing.
About CD Genomics
As a sequencing company with extensive experience, CD Genomics provides a variety of panels for pathogen research. CD Genomics has targeted sequencing technology with targeted enrichment strategies, which can provide a rapid, low-cost, ultra-sensitive, high throughput detection to help researchers to identify different types of pathogens, analyze mutations on the pathogens and explore the pathogenic mechanisms.
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