The the following Evidence Supporting Sanger Sequencing can be Considered as a SOTA Approach

Sanger sequencing, despite being a traditional method, still holds significant value in certain areas of genetic research and diagnostics. Here are some points that support its continued use and relevance:

1. High Accuracy: Sanger sequencing is known for its high accuracy, especially when used as an internal quality control method. It is less effective as a sole verification method for high-quality single-nucleotide polymorphisms, but its accuracy is undoubted.1

2. Widespread Use in Veterinary Diagnostics: The technique is extensively used in the veterinary diagnostic community for verifying PCR results and providing genetic sequence data for various analyses.2

3. Validation of Variants: Sanger sequencing is used to validate variants identified by next-generation sequencing (NGS). In one study, a small discrepancy was found between NGS and Sanger sequencing results, highlighting its role in confirming NGS findings.5

4. Specificity in Targeting DNA Regions: The method is effective in targeting specific regions of interest in template DNA using an oligonucleotide sequencing primer, which is crucial for focused genetic analysis.46

5. Optimal Results with Single Products: Sanger sequencing yields optimal results when the target DNA exhibits a single product, confirmed by electrophoresis, allowing for accurate sequencing of a homogeneous product.7

6. Efficiency in Monogenic Disorders: For disorders caused by variants in a single gene or a few genes, Sanger sequencing is considered time- and cost-efficient, making it a preferred method in certain genetic testing algorithms.8

7. Historical Significance: Developed in 1977 by Frederick Sanger, the method played a pivotal role in the sequencing of the first human genome, marking a significant milestone in genomic medicine.910

While Sanger sequencing may not be considered the state-of-the-art (SOTA) approach in all areas of genomics due to advancements in NGS technologies, it remains a valuable tool in specific applications, particularly where accuracy, cost-effectiveness, and targeted analysis are paramount. Its historical significance and continued use in certain fields underscore its ongoing relevance in the genomics toolkit.

What are the limitations of Sanger sequencing compared to next-generation sequencing (NGS) methods?

Sanger sequencing, while a robust and historically significant method, has several limitations when compared to next-generation sequencing (NGS) techniques. One of the primary limitations is the length of the DNA sequences it can effectively handle, which is generally shorter than what NGS can achieve1521. Additionally, Sanger sequencing is more expensive on a per-base basis, making it less cost-effective for large-scale projects1718. NGS platforms are known for their high-throughput capabilities, allowing for the sequencing of multiple samples in a single run, which reduces the overall cost per sample18. Furthermore, NGS technologies can simultaneously sequence more than 100 genes and whole genomes with low-input DNA, a capability that Sanger sequencing does not possess30.

How does the cost-effectiveness of Sanger sequencing compare to other sequencing methods for monogenic disorders?

Sanger sequencing is considered to be time- and cost-efficient for monogenic disorders, which are caused by variants in a single gene or a few genes8. It is particularly suitable for these types of disorders because it allows for targeted sequencing endeavors where the focus is on specific genes. However, when it comes to large-scale projects or the sequencing of multiple samples, NGS is generally more cost-effective due to its high-throughput nature1718. Sanger sequencing, on the other hand, may not be the best option for projects with limited budgets as it is more expensive per base1820.

What is the role of Sanger sequencing in the verification of PCR results and its contribution to phylogenetic analysis?

Sanger sequencing plays a significant role in the verification of PCR results and contributes to phylogenetic analysis by providing accurate genetic sequence data. It is widely used in the veterinary diagnostic community for this purpose2. The method is capable of determining the exact order of the four bases in a specific section of DNA, which is crucial for verifying PCR results and for phylogenetic studies2526. Furthermore, Sanger sequencing is used to provide the genetic sequence data needed for epidemiologic studies and forensic investigations, where accurate and reliable sequence information is essential2.

How does the accuracy of Sanger sequencing vary with different types of genetic variants or mutations?

Sanger sequencing is known for its high accuracy, which is particularly important when dealing with different types of genetic variants or mutations. It is especially suitable for verifying sequencing for site-directed mutagenesis or cloned inserts, with an accuracy of 99.99%30. The method is also vital in clinical analysis due to its ability to analyze genetic markers like SNPs and STRs, and to generate reliable sequence data31. However, the accuracy of Sanger sequencing can be influenced by the quality of the sample and the specific conditions under which the sequencing is performed. For optimal results, the target submitted for Sanger sequencing should exhibit a single product, as confirmed by capillary electrophoresis or gel electrophoresis procedures7.

What are the specific guidelines suggested by the Broad Institute for Sanger sequencing validation of variants identified by NGS?

The Broad Institute has suggested guidelines for the validation of variants identified by NGS using Sanger sequencing. Variants are to be validated if they meet certain criteria, such as having a minor allele frequency (MAF) less than 0.01 and an allele balance greater than 0.253436. These guidelines are commonly accepted as standard in the field and ensure that the Sanger sequencing validation is performed on variants that are likely to be genuine and relevant. The use of Sanger sequencing in this context serves as an additional quality control measure to confirm the presence of the variants identified by NGS.