Over 50 years ago, DNA sequencing development revolutionized the biological and medical sciences. Next Generation DNA Sequencing is the second modern revolution that has ushered in this dynamic field. Here’s what you should know before using the tool.
The field of genetic research is rather new. When we started from Mendel’s discoveries in the 1860s to the development of molecular biology in the 1950s, we have come a long way in understanding genetics. But during that time, we still couldn’t tap into the information contained in our DNA.
The science of genetics truly started to thrive in the early 1970s, with the development of Sanger’s chain-termination method- the first-generation sequencing.
In 1987, after the first automatic sequencing machine development, we truly witnessed a fast-paced and dynamic expansion in the field of genetics which has continued to evolve over the next 20 years.
The early 2000s mark the beginning of a new era. The development of sequencing by synthesis, also known as second-generation sequencing that allowed us to sequence millions of DNA fragments simultaneously.
It is now that we can confidently, and costs efficiently tackle whole-genome sequencing, resequencing and de novo genome sequencing, transcriptome, and epigenetics studies. With the revolutionary advancement in technologies, the whole genome sequencing cost has comparably come down.
The working of NGS
The workflow for a typical NGS has four steps:
Library preparation
In this step, Extracted DNA or cDNA are used as DNA samples. At both ends of the DNA fragments, Indexed adapter fragments are attached through reduced-cycle amplification. An adapter consists of a sequencing binding primer region, an index, and a unique sequence complementary to flow cell oligos at the end. This whole process enables the unequivocal assignment of the fragments to the correct sample in different concomitantly sequenced samples.
Amplification
In the second step, the double stranded DNA library is converted to a single-stranded library. These Seed DNA templates are used to produce a huge number of copies from each fragment of a single stranded DNA library forming a multiplexed pool of ssDNA libraries to form DNA cluster/nanoball. The process is simultaneously repeated for millions of DNA templates to form a library of DNA clusters/nanoballs, where each nanoball represents an individual seed DNA template. At the end of the cycle, the reverse strands are cleaved and washed away to prevent unwanted priming.
Sequencing
The third step is sequencing by Synthesis of complementary strands of the DNA on original template strand using fluorescently marked dNTP per cycle. Addition of each nucleotide complementary to the original template results in a Characteristic fluorescence signal. These signals are produced by millions of fragments undergoing synthesis simultaneously on a silicon chip (a.k.a. Flow cell) generating high intensity signal and therefore this process is also known as massively parallel sequencing. The length of the sequence fragments depends on the number of sequencing cycles. In case of Pair-end sequencing, forward strands are cleaved and removed at the end of first cycle, and the reverse strands are likewise sequenced.
Data analysis
In the quality control check process, adapter sequencing containing read (scores) less than 10% or 50% of the bases are removed. And these sequences are assigned to the correct library by identifying the unique indices present in the sequence. In the next process, stretches of base calls with the same read are locally clustered and paired to create contiguous sequences. These contiguous sequences are aligned and mapped back to the reference genome, further used for research studies and data analysis.
The next-generation DNA sequences are useful for detecting abnormalities across the entire genome, including substitutions, insertions, deletions, duplications, and copy number changes. The major strength of the next-generation sequences is that they can identify the abnormalities which were not identified before using other traditional techniques. The exome sequencing cost in India it’s also less expensive and has a faster turnaround time.
