So why Sequence?
- Throughout this class, we have been focusing on the phenotypes of the starters, but with DNA sequencing we can look at the genotypes of the microbial community by assessing which types of microbes are present in the control starter versus the starter with fruit in it. This will help us to discover if the type of fruit used, and the presence of fruit in general, affects the types of microbes present within the starter.
Previous Knowledge
- I have absolutely no previous knowledge of DNA sequencing, so this is all brand new to me.
Sanger sequencing vs. NGS
- The main difference between Sanger sequencing and NGS is that NGS can process millions of fragments at a time while Sanger sequence can only process a single fragment at a time. This causes NGS to be a far more efficient sequencing method as it is much faster in comparison.
NGS technology in GN 312
- The type of sequencing we will use in this class is called Illumina sequencing. The first step of this sequencing is sample prep where adapters are put on the end of the DNA fragments. The next step is cluster generation where oligos are used to form complementary strands on DNA fragments. These then form a bridge to the other fragments in bridge amplification. During sequencing, nucleotides are added onto the DNA fragment and light up with a specific color. All DNA fragments are sequenced simultaneously with this process. I did not really understand the sequencing step after this process. The final step is data analysis where forward and reverse reads are paired and grouped together using a gene library. These groupings are then compared to the reference genome to determine the genotype of the stands.
Citations
- https://emea.illumina.com/science/technology/next-generation-sequencing/ngs-vs-sanger-sequencing.html
- https://www.youtube.com/watch?v=fCd6B5HRaZ8