March 28, 2019

Rithvik Baratam

I. Title: Next Generation Sequencing and Metabarcoding

II. Rationale:

The purpose behind this lab was to use learn about Next Generation Sequencing and Metabarcoding. By learning the  Illumina sequencing, the process of determining a series of base pairs was enabled. In addition, we learned about the CyVerse program and its efficiency in dealing with large sets of DNA.

III. Materials:

• CyVerse Account
• Computer
• Projector

IV. Procedure:

Illumina Sequencing

1. DNA is first broken up into fragments sized 200 to 600 base pairs and adaptors are attached to the ends of DNA
2. DNA + adaptor fragments are then washed onto a flowcell.
3. Unlabelled nucleotide bases and DNA pol are added to lengthen the DNA strands such that they form a bridge between DNA fragments attached to the flowcell
4. Through heat, double-stranded DNA is broken down into single-stranded DNA
5. This results in clusters of identical strands of DNA
6. Fluorescent terminators and primers are added to the flowcell
7. Primers attach to the DNA sequenced.
8. DNA pol binds to the primers and adds fluorescently labeled terminators to new strand. Once added the terminator is removed.
9. Lasers scan flowcell to read fluorescent tags on nucleotides. First fluorescent groups are removed so next fluorescent groups can be added
10. Lasers then continue to scan flowcell to read fluorescent tags on nucleotides making the process accurate and efficient

V. Poster:

Final Draft of Poster

VI. Conclusion:

This lab gave us a better insight into the exact process of Next Generation Sequencing. The animations showed in class were helpful as they gave us a visual understanding as to how the process was carried out. In the future, as a class, we will choose a few DNA samples to send to get sequenced..