Erick Cornejo

3/29/2019

Objective and Purpose

The purpose of the lab was to introduce ourselves to next generation sequencing and to continue to talk about our metabarcoding. Next generation sequencing will play an important role in our data analysis and metabarcoding.

Procedure

Sequencing

1. Prepare Genomic DNA sample: DNA samples should be broken up and mixed with ligate adapter.
2. Attach DNA to surface: DNA Fragments should then be binded to the surface of the flow cell.
3. Bridge Amplification: Unlabeled Nucleotides and enzymes will be added. Bridge Amplification will take place on the surface of the flow cell.
4. Fragments become double stranded: At this stage, enzymes will begin to construct double stranded DNA through the incorporation of the unlabeled nucleotides.
5. Denature the double stranded molecules: Denaturization of the double stranded DNA will then result in single stranded DNA templates which will allow for further cloning.
6. Complete Amplification: Once Cloning of DNA is completed, a cluster will form.
7. Determine First Base: Add 4 labeled reversible terminations, primers, and polymerase to the flow cell. Conduct laser excitation.
8. Image First Base: Fluorescence should be emitted and captured into an image. Record findings.
9. Determine Second Base: Add 4 labeled reversible terminations, primers, and polymerase to the flow cell. Conduct laser excitation.
10. Image Second Chemistry Cycle: Fluorescence should be emitted and captured into an image. Record findings.
11. Sequence Reads Over Multiple Chemistry Cycles: Repeat cycles of sequencing to determine the sequence of bases in a given fragment.
12. Align Data: Compare all of the data in a data analysis.

Conclusion

In conclusion, through active learning methods, we instantly had a better understanding of next generation sequencing. This method of metacognition helped to break down what could be thought of as a long and complex cycle into something more imaginable and understandable. In the future, I hope to be able to conduct this procedure.

Objective

The objective for this lab was to research and learn about how DNA is analyzed and sequenced through Illumina sequencing.

Purpose

The purpose of this lab was to develop an understanding of the process of Illumina sequencing and whatgoes on in each individual step.

Procedure

We arrived in the computer lab and were given a brief mini-lecture about next-generation sequencing, its history, and its current applications to our work. Then we did a card game where we had to put the steps of Illumina sequencing in chronological order. Finally, we did our QTM’s using the knowledge we had gained from our time in lab.

Data

Steps for Illumina Sequencing

1. Fragment DNA and add adaptors so it can attach to the flow cell and amplify
2.  After amplification, sequence the DNA using reversible terminators
3. Sort each unique sequence and align to a database

Conclusion

This week’s lab was highly informative and, though the material was at some times difficult to understand, I feel like I have a much better understanding of the next steps of our research.

Myrnalid Zapata

03.29.19

Purpose: Learn how DNA sequences are produce and analyzed from the tube of extracted DNA, and explain the process of generation “reads” using Illumina sequencing. Set up a CyVerse account.

Procedure:

We were asked to put text cards in the correct order which describes the process of Illumina sequencing:

1. Break up genomic DNA into 200 to 6000 base pairs by using enzymes or by PCR
2. Denature  the double stranded molecules into single stranded molecules that have the adaptor and primer binding site.
3. Attach the DNA fragments to the flowcell through complementary binding of the adaptor to the oligos (primers) on the surface of the flowcell.
4. Replicate the DNA attached to form small clusters of DNA with same sequences.
5. Unlabelled nucleotides bases and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flowcell. This creates ‘bridges’ of double-stranded DNA between the primers on the flowcell surface.
6. The double-stranded DNA is then broken down into single-stranded DNA using heat, leaving several million dense clusters of identical DNA sequences.
7. Fluorescently labelled terminators are added to the flowcell.
8. The primer attaches to the DNA being sequenced.
9. The DNA polymerase then binds to the primer and adds the first florescently-labelled 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.
10. Lasers are passed over the flowcell to activate the fluorescent label on the nucleotide base.
11. The fluorescently-labelled terminator group is then removed from the first base and the next fluorescently-labelled terminator base can be added alongside.

Future Goals: Next week we are going to be working with the cloud computing/jupyter notebooks.

Conclusion: We learn that Next Generation Sequencing is a technology that is used throughout the sciences for analyzing single genomes and communities. This technique is built on the basics of PCR and sequence alignment. First Amplify, then Sequence and finally Analyze

Objective:

The goal of lab today was to learn more about the illumina sequencing procedure and familiarize ourselves with vocabulary related to the illumina sequencing procedure.

Procedure: 

Today, we were paired off into groups of two and given a bag with several cards that outlined the procedure of the illumina sequencing process. We spent about twenty to thirty minutes reviewing the powerpoint that outlined the procedure and used the powerpoint to play the cards and their corresponding images in the correct order. Then, we used our acquired knowledge to complete the QTM for this weeks lab.

Illumina Sequencing:

1. Break up the DNA into fragments around 200-600 base pairs.
2. Adaptors are attached to the DNA fragments.
3. Fragments are attached to the adaptors and are made single stranded.
4. DNA fragments are washed across the flowcell .Complementary DNA binds to the primers on to surface of the flowcell and unattached DNA is washed away.
5. DNA attached to the flowcell is replicated and forms small clusters of same sequence DNA. These clusters emit a signal that is able to be detected by camera.
6. Nucleotide bases and DNA polymerase are added to lengthen and join the strands of DNA attached to the flowcell.
7. Double stranded DNA is broken down into a single strand of DNA using heat.
8. Primers and nucleotides with terminators are added to the flowcell.
9. The primer attaches to the DNA being sequenced.
10. DNA polymerase binds to the primer and adds fluorescently labelled terminator to the DNA strand. No more bases can be added to the strand until the terminator base has been cut from the DNA.
11. Lasers pass over the flowcell to activate the fluorescent label on the nucleotide base. This is detected by a camera and recorded on a “read” by a computer.
12. The terminator group is removed from the first base and the next terminator can be added. This process is repeated until millions of clusters have been sequenced.

Data: 

Cyverse username: lesliemorales

Conclusion/Future Goals: 

Today in lab were we able to work and interact with people outside of our normal group. This allowed me, personally, to deviate from the normal way I do work with my group and learn how to adjust to working with others. I enjoyed doing the illumina card challenge because reading and rereading the steps until I understood the procedure allowed me to develop a better understanding of what was occurring. In the future, we will continue to learn about cloud computing.