The main steps of next generation sequencing are library preparation, sequencing, and data analysis. The choices you make at each step will affect 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®, Pacific Biosciences, and Oxford Nanopore Technologies.
During library prep, the nucleic acids are fragmented into shorter sequences (to combat sequencing bias) 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.
The third step in the NGS workflow is sequencing the libraries prepared in step two. There are many types of sequencing methods. The most popular method compatible with IDT products is provided by Illumina.
Illumina’s method is called sequencing-by-synthesis, in which fluorescently tagged bases emit a signal as they are added to a nucleic acid chain during synthesis. Illumina sequencing provides the highest throughput, largest data output, and lowest reagent costs.
Not sequencing with Illumina? We also help customers design custom solutions for other sequencing platforms. Contact us today to learn more about custom solutions.
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.
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.
This detailed overview walks you through major advances in capture and enrichment technology, types of targeted next generation sequencing, their applications, and more.