Advantages and limitations of next‐generation sequencing technologies: A comparison of electrophoresis and non‐electrophoresis methods

Abstract

The reference human genome provides an adequate basis for biological researchers to study the relationship between genotype and the associated phenotypes, but a large push is underway to sequence many more genomes to determine the role of various specificities among different individuals that control these relationships and to enable the use of human genome data for personalized and preventative healthcare. The current electrophoretic methodology for sequencing an entire mammalian genome, which includes standard molecular biology techniques for genomic sample preparation and the separation of DNA fragments using capillary array electrophoresis, remains far too expensive ($5 million) to make genome sequencing ubiquitous. The National Human Genome Research Institute has put forth goals to reduce the cost of human genome sequencing to $100,000 in the short term and $1000 in the long term to spur the innovative development of technologies that will permit the routine sequencing of human genomes for use as a diagnostic tool for disease. Since the announcement of these goals, several companies have developed and released new, non-electrophoresis-based sequencing instruments that enable massive throughput in the gathering of genomic information. In this review, we discuss the advantages and limitations of these new, massively parallel sequencers and compare them with the currently developing next generation of electrophoresis-based genetic analysis platforms, specifically microchip electrophoresis devices, in the context of three distinct types of genetic analysis.

Key Principles and Clinical Applications of “Next-Generation” DNA Sequencing

“…Throughput is a function of sequencing reaction time, the number of sequencing reactions that can be run in parallel, and lengths of sequences read by each reaction. The requirement for electrophoretic separation of DNA fragments for reading DNA sequence content in Sanger-based sequencing is the primary bottleneck for throughput with this method, increasing time and limiting the number of reactions that can be run in parallel (13). Despite efficient automation, each Sanger instrument can only read 96 reactions in parallel, and this restricts the technology's throughput to approximately 115 kb/day (1,000 bp; ref.…”

“…14). Current estimates suggest a cost of approximately $5 to 30 million USD to sequence an entire human genome using Sanger-based methods, and on one machine, it would take around 60 years to accomplish this task (8,13). Together, these cost and time constraints limit access to and application of genome sequencing efforts on this platform.…”

High Throughput Sequencing Advances and Future Challenges

“…Subsequently denaturation and cleanup of free moving enzyme, primers and the nucleotides, while the resulting molecules are arranged by their molecular weight (analogous of the point of termination) and the markers involved to the terminating ddNTPs is read out successively in order formed by the categorization step. With the application of existing Sanger sequencing techniques, it is precisely capable of 384 sequences [23] ranging from 600 to 1,000 nt, in length [24,25] though, the present 384-capillary systems are exceptional. The additional ordinary 96-capillary apparatus produce a utmost of more or less 6 Mb of DNA sequence apiece date, with expenses for consumables cost about $500/1 Mb.…”

Next Generation Sequencing and Cancer

“…Being cheaper and more advanced, NGS have shaped the new technical advances in different areas of cancer biology and treatment; moreover, massive sequencing platforms are more useful than other sequencing techniques. Several technical limitations of NGS can be including: quality control, data management and storage, reporting complex results and patent infringement that may affect laboratories using this technique [8,9,10].…”