SNPs and Human Diseases
SNPs, single nucleotide polymorphism, is the simplest genetic variation and occurs in every 1000 bases in human genomic DNA. Among the 3.5 million SNPs, some lie in intergenic regions (non-coding regions that play regulatory roles) while others are missense or silent mutations. These SNPs are related to diseases, drug response and sequence evolution.
Only a small fraction of SNPs are identified that are related to diseases affected by one or a few genes. However, with the advancement of genomic sequencing technologies, more and more SNPs or variants in multiple genes are found to be associated with complex heterogeneous disorders such as high blood pressure, diabetes and cardiovascular diseases. Of course, there are other disease-causing variations beyond SNPs, such as deletions, insertions, copy number variations (CNV) or structural variations.
Sanger sequencing for single or a few gene variants
Sanger sequencing is best used for diagnosis of diseases caused by single or a few gene SNP variants. As the gold standard for DNA sequencing, Sanger sequencing provides 99.99% accuracy and are routinely used for identifications of these variants with low cost and short turnaround time (see an example in this book chapter). NGS is only justified when a sufficient number of test orders come in to test a few genes in this category.
Disease-Targeted Sequencing for variant gene panels
For diagnosis of diseases that involve multiple known genes and hotspots or targeted drug therapies, a gene panel including a few genes to hundreds of genes is designed for cost-effective targeted NGS testing. The target sequencing provides great sequencing depth of coverage compared to sequencing the whole exome or genome (WES), thereby increasing test sensitivity and specificity. For this category, Sanger sequencing or WES is not a good choice compared to targeted NGS. The FDA has approved a few NGS gene panel tests for tumour profiling since 2017.
Sequence gaps produced in targeted NGS sequencing, if any, can be filled by Sanger sequencing, as suggested in the comprehensive ACMG (American College of Medical Genetics and Genomics) clinical laboratory standards for next-generation sequencing.
WES and Sanger sequencing for heterogeneous genetic disease studies
Although mainly used in research discovery, the whole-exome sequencing using NGS has a great clinical application potential and about 20% of diseases are diagnosed by WES. When targeted sequencing is not available, or it does not explain the patient’s phenotype, WES is a good choice. For example, in heterogeneous inherited genetic disease studies, WES is often used to sequence both affected and unaffected individuals in a family or its relatives to identify new genes associated with the disease. After variant filtering through bioinformatics tools, only a few gene variant candidates are prioritized for further segregation analysis, which involves variant confirmation by Sanger sequencing. Eventually, a conclusion can be made if a variant gene is involved in causing the disorder.