Scientists continue searching for the best ways to enhance Ribonucleic acid (RNA) sequencing and other vital bioinformatics techniques, and next generation sequencing (NGS) proves a highly effective methodology.
What Is Next Generation Sequencing?
The primary goal of NGS is to support the fragmenting of Deoxyribonucleic acid (DNA) and RNA into multiple pieces. In NGS, scientists add adapters and sequence the libraries to reassemble the DNA or RNA to form a specific genomic sequence. The concept of NGS is similar to capillary electrophoresis, which is “an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage.”
NGS empowers scientific researchers in their efforts to sequence thousands, or even millions, of DNA molecules at once, making it a faster, more accurate and cost-effective methodology. Since the technique offers a high-throughput sequencing (HTS) option and high-capacity sequencing capacity, it is an essential tool in clinical diagnostics, genetic disease research and personalized medicine.
There are three essential steps in NGS:
- Library Preparation. While sometimes time-consuming and cumbersome, it is a crucial component to the success of NGS workflow. It prepares DNA samples to become compatible with a sequencer. Scientists usually prepare libraries by fragmenting DNA and adding customized adapters at both ends of the strand.
- Sequencing. Scientists load the prepared libraries into a flow cell during this essential step, then place them on the sequencer. The scientist then performs a process called cluster generation that results in millions of copies.
- Data Analysis. Once the sequencing is complete, the process software identifies nucleotides through a process called base calling. It also offers a predicted accuracy of the base calls. Scientists typically sequence data into a standard data analysis tool or establish their data channel. There are now multiple available intuitive data apps to analyze NGS data without formal bioinformatics education or training.
What Is RNA Sequencing—What Are the Goals of This Process?
RNA sequencing (RNA-Seq) offers a revolutionary approach to studying the transcriptome. Science Direct describes the transcriptome as “one measure of the cellular status.” Thanks to the ease of genome-wide profiling through sequencing technologies, transcriptome analysis has become a critical part of bioinformatics.
RNA-Seq serves as a sensitive and accurate tool to measure the transcriptome, providing scientists and researchers with visibility and insight into previously undetected or indiscernible changes occurring in disease states in response to:
- Various environmental conditions
- A broad range of other study designs and conditions
RNA-Seq through NGS offers researchers better coverage and more resolution of the dynamic aspects of the transcriptome than the previous microarray-based and Sanger sequencing methods.
In NGS RNA-Seq, scientists can explore new transcripts, identify alternatively spliced genes, and detect allele-specific gene expression.
It is crucial in all RNA-Seq experiments that scientists prepare a library of cDNA fragments attached to adapters to synthesize for successful sequencing.
During processes in molecular biology, DNA becomes transcribed into multiple copies of messenger RNA (mRNA). At that point, they become proteins, allowing cells to produce multiple protein molecules from that single gene.
Next generation sequencing is a significant factor in improving RNA sequencing, providing a broad and dynamic range and enhanced sensitivity that offers better detection of differentially expressed genes and quantifiable data within the transcriptome.