NGS (next generation sequencing) is a technique for identifying the sequence of DNA or RNA in order to investigate genetic variation linked to illnesses or other biological phenomena. First commercially available in 2005, this method was named “massively parallel sequencing” because it allowed the sequencing of several DNA strands at once, rather than one at a time as with capillary electrophoresis (CE) in standard Sanger sequencing.
Next-Generation Sequencing Workflow
Regardless of the instrument technology utilized, a typical NGS experiment follows the same stages.
1. Construction of Library
The sample will be used to start a sequencing library. Double-stranded, short fragments of between 100 and 800 bp are created from the DNA (or cDNA) sample. Fragmentation of the DNA can be achieved through a variety of methods, including physical shearing, enzyme digestion, and PCR-based amplification of particular genetic areas, depending on the application. The fragment library is then formed via ligation of the DNA fragments to technology-specific adaptor sequences. Each sample may be marked with a unique DNA sequence if these adaptors have a distinct molecular “barcode.” Multiple samples can be combined and sequenced at the same time using this method. This method, also commonly referred to as “multiplexing” or “pooling,” saves both time and money during sequencing investigations while also controlling for workflow variance.
Two other methods of library preparation, in addition to fragment libraries, are paired-end libraries and mate-pair libraries. Paired-end libraries allow users to sequence the DNA fragment from both ends, rather than the traditional single-direction sequencing. Paired-end libraries are made in the same way as ordinary fragment libraries, but they feature adaptor tags on both ends of the DNA insert, allowing two-way sequencing. This approach makes mapping reads easier and may be used to discover genomic rearrangements, repetitive sequence elements, RNA gene fusions, and splice variants.
2. Clonal Amplification
The DNA library must be mounted to a solid surface and clonally amplified before sequencing to enhance the signal that can be detected from each target. From there, each DNA molecule in the library is attached to the surface of a bead or flow-cell and PCR amplified to generate a collection of identical clones throughout this procedure.
3. Sequence Library Creation
A sequencing apparatus is used to sequence all of the DNA in the library at the same time. Despite the fact that each NGS technique is different, they all use a variant of the “sequencing by synthesis” process, which involves reading individual bases as they expand along a polymerized strand. This is a cycle containing the following steps: synthesis of the single-stranded DNA base, detection of the integrated base, and finally removal of reactants to restart the cycle.
4. Data Analysis
The three stages of data analysis in next generation sequencing are: primary, secondary, and tertiary. The transformation of raw signals from instrument detectors into digital data or base calls is known as the primary analysis. During the cycle of sequencing, raw data is collected. Files containing base cells are integrated into sequencing reads called FASTQ files. The corresponding quality ratings result from the primary analysis, called the Phred quality score. The secondary analysis includes read quality filtering and trimming, followed by reading alignment to a reference genome or read assembly for new genomes, and ultimately variant calling.
NGS analysis is often conducted by bioinformatics professionals due to the complexity of NGS data and accompanying algorithms. However, without bioinformatics skills or extra lab personnel, you can now examine NGS data using straightforward data analysis software. Data visualization, variant calling, sequence alignment, and interpretation are all services provided by these technologies.
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