NGS, or next-generation sequencing, is in the news more and more often. This method of genetic sequencing is faster, more efficient, and more precise than previous methods. Therefore, labs are rushing to learn how to do it so that they, too, can benefit from it.
While the details of genetics are complex, it’s not hard for a lab with qualified people to start using NGS in its own research. Read on to learn the basics of next generation sequencing.
A Quick Overview
The basic process involves fragmenting DNA or RNA into multiple pieces. Adapters are added to each piece so that the sequencer knows where the pieces begin and end. The libraries are then sequenced, and finally, the results are put together to get the whole genomic sequence.
This is similar to capillary electrophoresis in principle, but there is a critical difference: Next generation sequencing handles millions of fragments in a massively parallel way, which improves speed and accuracy while reducing costs.
Preparing for Your Workflow
The first step is to isolate and purify your nucleic acid sample(s). Be aware that some DNA extraction methods introduce inhibitors, so take care to use a protocol that has been properly optimized for your sample type. If sequencing RNA, convert it to cDNA using reverse transcription. Finally, do quality control on your sample. Use UV spectrophotometry to asses purity, and fluorometric methods for quantitiation of the nucleic acid.
Step 1: Library Prep
This is crucial to the success of your NGS efforts. In it, you prepare your DNA or RNA samples to be compatible with your sequencer. Once prepared, the results are called “libraries.” Typically, preparation is done by fragmenting the nucleic acid and adding specialized adapters to each end of the fragments. Adapters have complementary sequences that let the fragments bind to the flow cell. Once this is done, the fragments can be amplified and purified.
It is common to pool multiple libraries together for one sequencing run. This is known as multiplexing, and saves resources. When multiplexing, unique index sequences are added to each library so that they can be distinguished during data analysis later on.
Step 2: Sequencing
Now, libraries are loaded onto the flow cell and placed into the sequencing machine. In a process known as cluster generation, the libraries are amplified until there are millions of copies of single-stranded DNA. Some sequencers do this automatically.
Step 3: Data Analysis
Once the actual sequencing is complete, the sequencing instrument’s software identifies nucleotides in a process known as “base calling.” The software will also predict the accuracy of its own base calls. You can also import the data into a standard analysis tool or use your own custom pipeline.
Data analysis is easier now than it has ever been. There are apps that allow the analysis of NGS data without the need for bioinformatics training or even extra lab staff. Apps exist that provide variant calling, sequence alignment, data visualization, and even interpretation.
As this shows, next generation sequencing is no longer limited to the largest or most financially-endowed research centers. It can be done by a wide variety of laboratories, and software exists to streamline the process and handle many data analysis functions.