The next generation sequencing workflow consists of four key steps: nucleic acid extraction, library preparation, sequencing, and analysis. The choices you make at each step are critical for your experimental outcomes. Learn more about each step below.
NGS starts with genomic material with either DNA or RNA that is extracted from cells or tissue. Your choice of isolation method or kit is critical to ensure proper lysis of cells and tissues so that you can capture the genetic material in the sample. Selecting the right isolation method or kit will maximize nucleic acid yield, purity, and quality for the next step of NGS library preparation. You should confirm the yield, purity, and quality of your sequences before moving on to the library preparation step.
The next step in the NGS workflow is library preparation and provides everything you need to do to your isolated, purified genomic material for sequencing. IDT products are compatible with many types of sequencing platforms, including Illumina®, Ion Torrent, MGI, Element Biosciences, Singular Genomics, Ultima Genomics, Pacific Biosciences, and Oxford Nanopore Technologies,
During library prep, nucleic acids are typically fragmented into shorter sequences and adapters are attached to the sequences to make them compatible with the sequencer. Unique identifying sequences called “barcodes” or “indexes” are added to the samples at this stage. Barcodes allow samples to be combined, which is referred to as “pooling” or “multiplexing.” This allows the researcher to perform sequencing simultaneously which saves time and money.
An alternative to whole genome sequencing (WGS) is targeted sequencing, where specific portions of the genome, such as marker genes or other genomic regions of interest, are sequenced in lieu of the entire genome. This approach, called enrichment, is often faster and more cost-effective than sequencing the entire genome. Enrichment can be performed either during library prep (amplicon sequencing) or after library prep (hybridization capture), depending on the enrichment method chosen.
This detailed overview walks you through major advances in capture and enrichment technology, types of targeted next generation sequencing, their applications, and more.
The last step in the NGS workflow is analyzing the sequence reads produced by the sequencer. Just as there are many sequencing methods, there are many data analysis approaches, but they generally fall into one of two categories: read processing and sequence analysis.