Technologies for DNA and RNA sequencing are crucial to unlock secrets held within our genome and transcripts that can be used to better understand biology and impact medicine. Sanger sequencing is traditionally being used to sequence oligonucleotide strands one at a time by capillary electrophoresis. While this method is still considered the gold standard for analyzing small numbers of gene targets and samples, next-generation sequencing (NGS) is quickly overtaking it.
NGS, also known as massively-parallel sequencing, enables the interrogation of larger numbers of genes with high throughput and low cost per run. Continual improvements have also enabled NGS to capture a broader spectrum of mutations and be more accurate in detecting variants at low allele frequencies without bias.
There are a few main companies providing instruments for NGS, but their workflow is similar. The first step is library construction where DNA or complementary(c) DNA generated from RNA is fragmented and bound to adaptors that have a unique molecular “barcode.” With this barcode, each fragment is uniquely labeled, and multiple samples can be pooled together to save time during sequencing. The second step is clonal amplification, where more copies of each DNA fragment are amplified via polymerase chain reaction. The third step is sequencing, where most instruments use optical signals to assess nucleotide incorporation during DNA synthesis. The final step is analysis.
NGS to boost microbiome research
The microbiome describes the interactions of microorganisms including bacteria, fungi, and viruses with their ecological environment, such as a human body. Such interactions can be further broken down into commensal, symbiotic, and pathogenic.
A variety of bacteria resides in our guts and influence our health, such as through adsorption and secretion of metabolites. NGS has been useful for taxonomical identification and classification of the microbiome in different parts of our bodies, including the intestinal and respiratory tracts. Kasai and colleagues studied the composition of gut microbiota in Japanese subjects using NGS technology and found that there were significant differences in the bacterial species. For instance, there was higher bacterial species diversity in obese individuals.
NGS technologies have also facilitated an extension of such research to evaluate the effects of probiotics to modulate perturbed microbiota. Suez and co-workers found that probiotics supplementation might be ineffective as resident gut bacteria can resist the mucosal presence of probiotics strains. Probiotics supplementation was only useful in a subset of individuals, suggesting that this is likely not a universal approach to influence gut microbiota composition and that personalized probiotics development should be further studied.
Antibiotics can also significantly alter the microbiome and play a role in disease onset and progression. Using NGS technologies, scientists have experimentally identified bacteria species that are affected by antibiotics and how their absence correlates with changes in immunity and disease susceptibility. Specifically, researchers have found that the use of antibiotics can have negative impact on gut microbiota by reducing species diversity (causing loss of bacterial ligands recognized by host immune cells), changing metabolic activities and selection of antibiotic-resistant organisms that can cause recurrent Clostridioides difficile infections.