Are you designing a new experiment that involves DNA sequencing? In that case, one of the first questions that pop into your mind is likely to be “which DNA sequencing method should we use?” They are similar since both of them sequence DNA fragments. Before the 90s, Sanger sequencing was the go-to method of DNA sequencing. As the demand for large-scale DNA sequencing became more prominent, Next-generation Sequencing (NGS) methods began to gain popularity.
Sanger sequencing vs. NGS: an Overview
Sanger sequencing has been hailed as the gold standard for small scale DNA sequencing with 99.9% accuracy rates. It is quite effective in the determination of DNA mutations. A single run produces only one forward, and one reverse read. However, Sanger sequencing is incapable of running parallel sequencing reactions. At the same time, it has limited sensitivity. Detecting somatic cancer cell mutations using Sanger sequencing techniques is quite challenging since these tumors can be highly heterogeneous and interspersed with healthy tissue.
On the other hand, NGS can run massively parallel DNA sequencing reactions. They can sequence hundreds of thousands of small, medium, and large-sized DNA fragments simultaneously. It can sequence millions of fragments depending on the experimental setup. It is fast and cost-effective. NGS software has made DNA sequencing affordable and quick for almost all research laboratories across the globe.
What are the Basic steps of Sanger Sequencing Technique?
The Sanger sequencing method is a chain-termination method. In the early years, it used a dye-labeled dideoxynucleotide to terminate the DNA elongation. The four labeled dideoxynucleotide molecules bound to complementary bases in the template strands and terminated the elongation reaction.
After the reaction stopped, the resulting fragments of DNA are subjected to capillary gel electrophoresis. That would separate the DNA molecules by their size. The differently labeled ddNTPs would be picked up by the detector complete with their relative positions.
The chromatogram generated after the analysis has the distinct peaks representing the sequence of the incorporation of the ddNTPs in the sequencing reaction. It is quite the straightforward and reliable process, as long as the target DNA is small to medium (up to 700-900 base pairs) in size. Due to the working principle of Sanger sequencing, it is quite time-consuming to complete the sequence of multiple large DNA fragments.
Why do researchers and clinicians Prefer NGS?
On the other hand, NGS is the second generation of DNA sequencing techniques. It is a high throughput technique that can sequence millions of DNA fragments at one go. It takes very less time when coupled with automated NGS software as compared to the Sanger sequencing method. NGS refers to a variety of high throughput techniques that capitalize on different sequencing approaches.
The NGS approach provides a tremendous bulk of sequencing information per run. Its ability to generate sequence information from multiple fragments on a microscale makes NGS techniques popular among teams running large scale DNA sequencing reactions. While, almost 20 years ago, sequencing the human genome took more than $3 billion and 13 years for completion, using automated NGS software, it now takes lesser than $1500 to achieve the same.
What contributes to the high-speed and cost-efficiency of NGS?
So, where do the high speed and affordability of NGS technology stem from? If you have used NGS technology in your experiments before, you must have noticed that there are specific working principles and technical steps that set NGS apart from Sanger sequencing. The use of automated NGS software adds more credibility and reproducibility to the DNA seq analysis reports.
Here’s a step-by-step breakdown of a general NGS DNA sequencing procedure –
- The preparation of a DNA library
The target DNA strand is broken down, and the DNA adapters of known sequences are ligated to the DNA. These bind the DNA to the support. The resulting library is amplified before sequencing the DNA fragments. In the next steps, this library is used as a template.
- Generation of cluster
The library is loaded into the flow cell for cluster generation. In this step, the DNA fragments are captured by support-bound oligo-nucleotides. The template preparation is deemed complete when the cluster generation is complete.
- Sequencing techniques
The sequencing techniques can vary significantly depending upon the proprietary technology used by individual companies. Some use reversible terminator-based methods, while others might rely on hybrid sequencing techniques.
- Data analysis
During the analysis and alignment of DNA sequencing data, the NGS software aligns the identified reads to a reference genome. Depending on the aim of the experiment, one can opt for one or more variants of the analysis, including the identification of SNP, IN/DEL mutations, or metagenomic analysis.
What are the Advantages of NGS?
NGS methods offer several distinct advantages over Sanger sequencing. Here are some of the benefits you should know before choosing the best-fitting sequencing technique for your experiment –
- NGS has higher sensitivity, and it can easily detect low-frequency variants
- They have a faster turnaround time for a high volume of sample DNA
- This method has a lower limit of detection
- It offers comprehensive genomic coverage
- It can be modified for whole-genome sequencing, whole-exome sequencing and targeted sequencing of genes
- NGS methods provide higher throughput with multiplexing of samples
- It can sequence hundreds and thousands of genes simultaneously
- It is much cheaper than Sanger sequencing for large sample volumes
Due to its customizable and flexible nature, NGS technologies can now be used in the fields of phylogenetic studies, metagenomics, epigenetic research, and transcriptome analysis. Today, NGS has become indispensable for the determination of novel variants and pathogen subtyping.
What should you never forget while using an NGS technique?
The quality of analysis of the sequence reads plays a significant role in the superiority of the results. With a wide variety of NGS techniques now available for the sequencing of DNA and RNA, you need to ensure that you are choosing an NGS platform that offers you the whole deal. You should have access to automated NGS software that offers reproducible and high-accuracy sequence analysis. The competent NGS software should provide you with QC plots, read counts, genome and variant browsers, variant summary reports, and coverage plot. Latest NGS analysis software offers to set up WGS and WES analysis within a couple of minutes with adjustable parameters.