The use of high-throughput next-generation sequencing (NGS) technologies continues to provide a wealth of sequence data, resulting in significant advances in a wide range of research areas and applications. Key among these is liquid biopsy – the ability to sequence nucleic acids found in circulation will enable scientists to identify key mutations and more specifically distinguish closely related ncRNAs.
Precision and reliability of NGS results depends on the ability to quickly and accurately obtain data from complex samples, and is dependent on technologies that provide sufficient yields of targeted, high-quality nucleic acids. The most common challenges experienced by researchers along the NGS workflow are accessibility of sequence information from clinically relevant samples and the complexity of subsequent data analysis and interpretation.
Generating reliable NGS data depends on the sample quality. Researchers need a diverse range of solutions that will allow them to deliver high yields of superior-quality nucleic acids and ensure outstanding results – on any sequencing platform. Researchers should be able to achieve high-quality DNA and RNA from challenging sample types, amplify the entire genome and transcriptome of single cells with minimal bias, and save time while standardizing the workflow with automated purification.
DNA sequencing technology has changed significantly in the past decade. At the beginning of this century, Sanger sequencing was the major technology. Sequencing the whole genome took a few years, but right now many human genomes can be done in one sequencing run with the latest sequencers. In the meantime, the cost of DNA sequencing has considerably dropped. Today the human genome can be sequenced for less than $5,000.
One of the methods used for amplification of the DNA library is called bridge PCR. The amplification is done on a flat surface called a flow cell that’s coated with two types of primers, corresponding to the adaptors. The amplification process occurs in cycles, with one end of each bridge tethered to the surface. Clusters of DNA molecules are generated on the flow cell, and each cluster is originated from a single DNA fragment. The number of DNA fragments used for cluster generation is critical. Too many fragments will result in too many clusters on a flow cell. The cluster then can be dense and overlap and the signal cannot be distinguished. Too few DNA fragments, on the other hand, will result in not enough clusters on a flow cell.
Another method of building the DNA library is called emulsion PCR. The solid surface used is a bead coated with one type of the primer corresponding to one of the adaptors. The other primer required is supplied in solution. Different DNA fragments as well as the bead are separated within a water droplet in oil. Each droplet ideally contains 1 DNA fragment and 1 bead. DNA molecules are synthesized on the bead. Each bead bears DNA originated from a single DNA fragment. After DNA amplification, the beads bearing DNA are collected and deposited into the wells of the sequencing chips, one well for one bead. In this system, too many DNA fragments in the system results in multiple DNA molecules in one droplet, while too few results in too many empty beads.
The complete NGS workflow includes sample preparation and the isolation of samples (DNA/RNA) which takes about several hours to days. The library construction phase prepares libraries for the specific platforms, lasting 4–8 hours. The subsequent sequencing run can last anywhere from 8 hours to several days. The last step is data analysis where the application of specific data is analyzed in the pipeline, requiring several hours to a few days. The advances that have led to quicker, less costly sequencing make NGS more accessible than ever. There is no doubt that its importance will continue to increase in translational research and clinical applications, including the development of accurate, reliable liquid biopsies.
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