Next-Generation Sequencing (NGS) offers a hypothesis-free approach that does not require prior knowledge of sequence information. NGS makes the simultaneous analysis of many genes possible and cost-effective. It is ideal for key stages in the workflow such as the acquisition of a cell line, banking and/or end of workflow.
Next-Generation Sequencing (NGS) technology has made whole genome sequencing (WGS) possible at an individual level. It allows the simultaneous analysis of many genes, which helps researchers understand the complexity of mutations in hPSCs.
The NGS workflow typically involves several key steps, including sample preparation, sequencing library construction, sequencing and data analysis. During sample preparation, the DNA is extracted from the biological sample then fragmented into smaller pieces. These fragments are converted into a sequencing library by adding adapters and undergoing amplification. Then, the prepared library is loaded into a high-throughput sequencing platform, where millions of DNA fragments are simultaneously sequenced. The sequencing data generated is then processed and analyzed using bioinformatic tools to align the reads, identify genetic variants and interpret the results. The NGS workflow has significantly accelerated genomic research, enabled personalized medicine and contributed to our understanding of complex genetic diseases.
Targeted NGS panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes.
For assay development, amplicon-based methods have been preferentially used for their shorter preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of its higher testing accuracy and reduced susceptibility to artifacts.
Sanger sequencing allows the analysis of a single, distinct sequence in each sequencing reaction. In contrast, NGS enables the simultaneous sequencing of thousands of reactions from multiple samples within a single reaction.
Consequently, NGS enables cost-effective screening of a large number of samples and the detection of multiple variants across targeted regions of the genome. This approach would be both costly and time-consuming if performed by Sanger sequencing.
Targeted sequencing offers several advantages over Whole Genome Sequencing (WGS). It is a cost-effective approach that focuses on specific regions of interest, resulting in reduced data generation, storage and analysis costs. Targeted sequencing achieves a higher depth of coverage in the targeted regions, improving sensitivity for variant detection. It is particularly useful for studying traits associated with specific genomic regions. The smaller data volume simplifies data management and analysis, enabling efficient variant calling and annotation. Targeted sequencing is scalable and suitable for large studies, providing a focused and cost-effective approach for investigating specific genomic regions.
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