Sydney Ortenberg

March 29, 2019

Lab 1o: Next Generation Sequencing & Metabarcoding

Objective/Purpose:

The objective of this lab was to introduce another type of sequencing; Next Generation Sequencing and Illumina. The purpose of this lab was to learn about other types of sequencing, what it does, and also learn about a large database for metagenomics called CyVerse.

Procedures:

1. Edit Poster prior to lab to turn in for final draft.
2. Assort Illumina steps in order using the playing cards with a partner.
3. Create a username for Cyverse and record on the class spreadsheet.
4. Complete QTM.

Data:

The chronological steps of Illumina sequencing:

1. Break up genomic DNA into more manageable fragments of around 200 to 600 base pairs by using enzymes or by PCR.
2. Tag the DNA fragments with short sequences of DNA called adaptors.
3. Denature the double-stranded molecules into single-stranded molecules that have the adaptor and primers binding site. This is done by incubating the fragments with sodium hydroxide.
4. Attach the DNA fragments to the flow cell through complementary binding of the adaptors to the oligos (primers) on the surface of the flow cell.
5. Replicate the DNA attached to the lyocell to form small clusters of DNA with the same sequence.
6. Unlabeled nucleotide bases and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flow cell. This creates ‘bridges’ of double-stranded DNA between the primers on the flow cell surface.
7. The double-stranded DNA is then broken down into single-stranded DNA using heat, leaving several million dense clusters of identical DNA sequences.
8. Primers and fluorescently labeled terminators (a version of nucleotide base- A, C, G, or T- that stop DNA synthesis) are added to the flow cell.
9. The primer attaches to the DNA being sequenced.
10. The DNA polymerase then binds to the primer and adds the first fluorescently-labeled terminator to the new DNA strand. Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA.
11. Lasers are passed over the flow cell to activate the fluorescent label on the nucleotide base. This fluorescence is detected by a camera and recorded on a computer. Each of the terminator bases (A, C, G, and T) give off a different color.
12. The fluorescently-labeled terminator group is removed from the first base and the next fluorescently-labeled terminator base can be added alongside. And so, the process continues until millions of clusters have been sequenced.

Storage: N/A since in computer lab.

Conclusion/ Future Steps: 

This lab introduced us to a different sequencing method called Next Generation Sequencing and we were able to become familiar with its methods so we can use it. Through the presentation of the history and sorting out the cards, we were able to see how the process worked. The Cyverse database allows scientists and researchers to analyze large amounts of data since most normal computers do not have the capacity to do so. In the future, I am looking forward to using Cyverse to analyze our data from this past semester and see how it all plays out. I think that Cyverse is a unique platform and we can learn a lot from it.