Overview of NGS approaches and methods
A novel technique for DNA and RNA sequencing as well as variant/mutation detection is called next-generation sequencing (NGS). NGS can quickly sequence an entire genome or hundreds of thousands of genes.
Next-generation sequencing (NGS) is a technology for determining the sequence of DNA or RNA to study genetic variation associated with diseases or other biological phenomena. Introduced for commercial use in 2005, this method was initially called “massively-parallel sequencing”, because it enabled the sequencing of many DNA strands at the same time, instead of one at a time as with traditional Sanger sequencing by capillary electrophoresis (CE). In the current genetic analysis context, each of these tools has a place. Sanger sequencing can be completed in a single day and is most effective when evaluating modest numbers of gene targets and samples.
Sanger sequencing is frequently used to validate NGS results because it is regarded as the gold standard for sequencing technology. Single nucleotide variants (SNVs), copy number and structural variants, and even RNA fusions are among the various genomic features that can be found and analyzed in a single sequencing run thanks to NGS. It also makes it possible to examine hundreds to thousands of genes simultaneously in multiple samples. The optimal throughput per run is made possible by NGS, allowing for rapid and economical study completion. NGS offers additional benefits over Sanger sequencing, such as reduced sample input needs, improved accuracy, and the capacity to identify mutations at lower allele frequencies. NGS’s speed, precision, and throughput have transformed genetic analysis and made it possible for new uses in reproductive health, environmental, agricultural, and forensic science as well as genomic and clinical research.
Steps in NGS workflow
A sequencing “library” must be constructed from the sample. The DNA (or cDNA) sample is divided into relatively short (100–800 bp) double-stranded segments. Depending on the specific application, DNA can be broken up using a variety of techniques, including physical shearing, enzyme digestion, and PCR-based amplification of specific genetic regions. The resulting DNA fragments are ligated to technology-specific adaptor sequences to generate a fragment library. These adaptors may also carry a unique molecular “barcode” that allows each sample to be uniquely identified by its DNA sequence. In order to tag each sample with a distinct DNA sequence, these adaptors might also carry a unique molecular “barcode.”
Apart from fragment libraries, paired-end libraries and mate-pair libraries are two further specialized library preparation techniques. Unlike standard sequencing, which only happens in one direction, paired-end libraries enable users to sequence the DNA fragment from both ends. Similar to standard fragment libraries, paired-end libraries are constructed with adapter tags on both ends of the DNA insert to allow for bidirectional sequencing. This approach facilitates read mapping and can be applied to enhance the identification of repeated sequence elements, RNA gene fusions, splice variants, and genomic rearrangements. However, single direction sequencing has also been able to discover these traits with to advancements in contemporary library preparation techniques and analytic tools. Mate-pair libraries require substantially bigger DNA inserts (more than 2 kb and up to 30 kb) and are more difficult to construct than fragment or paired-end libraries. Two reads that are distant to one another and in the opposite orientation are produced when mate-pair libraries are sequenced. Mate pair sequencing is helpful for de novo assembly, detecting significant structural variants, and identifying complicated genomic rearrangements by using the physical information shared by the two sequencing reads.
The DNA library needs to be clonally amplified and affixed to a solid surface before sequencing in order to enhance the signal that can be picked up from each target. Each distinct DNA molecule in the library is attached to the surface of a flow-cell or bead during this procedure, and it is then amplified using PCR to produce a collection of identical clones. In the case of Ion Torrent technology, library molecules are added to beads using a procedure known as “templating.”
All DNA in the library is sequenced simultaneously using a sequencing instrument. While NGS technologies vary, they all use a “sequencing by synthesis” method, which involves synthesizing DNA bases on a single strand, detecting the incorporated base, and then removing reactants to repeat the process. Most sequencing instruments use optical detection for nucleotide incorporation, while Ion Torrent instruments use electrical detection to sense the release of hydrogen ions during DNA synthesis.
NGS experiments produce large amounts of complex data with short DNA reads. While each platform has unique algorithms and tools, they follow a similar analysis pipeline and use common metrics to assess data quality. Analysis consists of three steps: primary, secondary, and tertiary. Primary analysis processes raw signals from detectors into digitized data, resulting in FASTQ files with base calls and quality scores. Secondary analysis includes read filtering, trimming, alignment to a reference genome, and variant calling, producing a BAM file with aligned reads. Tertiary analysis is the most complex, focusing on interpreting results and extracting meaningful information from the data.
NGS analysis is usually handled by bioinformatics specialists due to its complexity. To assist users without specialized training, platforms like Ion Torrent offer intuitive software that simplifies the analysis and eliminates the need for programming skills.
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