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giganticFISH Protocol: Channel Hunters - Gene Trees

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giganticFISH Protocol: Channel Hunters - Gene Trees

Introduction

The project is split into three labs over three weeks: WEEK 1 Species Tree RESEARCH LEVEL 1: Species Tree Vertebrate species tree are well-resolved. This work will generate a species tree that replicates what is already commonly known. It offers an opportunity to get your sea legs in phylogenomics research and will verify you have the tools and workflow working correctly. WEEK 2 Gene Tree RESEARCH LEVEL 2: TOP SECRET Gene Tree TOP SECRET gene evolution is well known in many animals but we discovered this summer highly unusual evolutionary patterns of TOP SECRET when looking across vertebrate diversity. Your work here represents critical replication of our gene tree findings to ensure things are what we think they are. TOP SECRET in toothed whales is a super exciting but brand new and still unpublished result - so TOP SECRET is TOP SECRET. The secret will be revealed. Please limit who you tell. Interviews on CNN not advised. WEEK 3 Gene Tree RESEARCH LEVEL 3: New Gene Tree There are over 20 known ion channels that respond to mechanical stimulation in human and may also respond to ultrasound. We have not - and no one in the scientific literature has ever - looked specifically at evolution of these channels in echolocating animals vs all major groups of vertebrates. Your own work in the lab will do produce gene trees for assigned ion channels and break new ground - we can't wait to see what you DISCOVER!!! MAJOR STEPS PER LEVEL A Build alignment B View alignment C Build Tree D View tree E Annotate tree F Post tree

Materials

giganticFISH website
- gigantic.fish
giganticFISH Slack workspace
- giganticFISH Slack workspace
Benchling giganticFISH Channel Hunters Protocol
- giganticFISH Benchling protocol
CIPRES individual account
- CIPRES Cyberinfrastructure for Phylogenetic Research
- DEMO video on how to work in CIPRES
Species Tree gene sets - Species-11 and Species-133
- Species detail (some links are dead but the page will be updated soon): https://gigantic.fish/genus-species-background-and-genomes/
- Species Tree - 11 species: https://gigantic.fish/wp-content/uploads/2020/11/giganticFISH-11_species-250_busco-genes-fasta.txt
- Species Tree - 133 species: https://gigantic.fish/wp-content/uploads/2020/11/giganticFISH-133_species-250_busco-genes-fasta.txt
- SPECIES-133 TREE FILE FOR iTOL: https://gigantic.fish/wp-content/uploads/2020/11/giganticFISH-133-tree.nexus_.txt
AlignmentViewer
- AlignmentViewer
- Trimmed MAFFT alignment of Species-133 Alignment: https://gigantic.fish/wp-content/uploads/2020/11/gigantic-trimmed.txt
TreeViewer: iTOL Interactive Tree of Life
- iTOL
- login giganticfish password g1g4nt1cf1sh
Xodo
- Xodo PDF Annotator
ANNOTATION PRACTICE: GENE TREE TRP SUPERFAMILY - DO THIS FIRST
- FASTA FILE - TRP Superfamily FASTA: https://gigantic.fish/wp-content/uploads/2020/12/trp-superfamily.fasta_.txt
ANNOTATION PRACTICE: GENE TREE LOLA TRPA1 SPECIES-127 - DO THIS SECOND
- TREE FILE - https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-TRPA1_X_species-127-AGS-treefile.txt
EXAMPLE ANNOTATED SPECIES-11 TREE
- https://gigantic.fish/wp-content/uploads/2020/11/species-tree-mini-skinny-anno.png
EXAMPLE ANNOTATED LOLA TRPA1 SPECIES-127 TREE
- https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-TRPA1_X_species-127-AGS-fasta.pdf

Procedure

START HERE:

Determine your gene based on the table below.

Level 3

	A A	B B	C C	D D	E E	F F	G G	H H	I I	J J	K K	L L
1 1	iTOL Team	Team	People 1-3	Gene Symbol	Gene Name	FASTA FILES FOR CIPRES	TREE FILES FOR iTOL IF CIPRES IS SLOW OR THROWS ERRORS	Gene Page	Background	Background	Background	Source Species
2 2	T01	Team HS1	Quynh-Vi, Marbely, Alicia	GRIN1	Glutamate receptor ionotropic, NMDA 1	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN1_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN1_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q05586	https://en.wikipedia.org/wiki/GRIN1	https://en.wikipedia.org/wiki/Glutamate_receptor	https://en.wikipedia.org/wiki/NMDA_receptor	human
3 3	T02	Team HS2	Fatima, Noah, Christian	GRIN2A	Glutamate receptor ionotropic, NMDA 2A	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN2A_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN2A_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q12879	https://en.wikipedia.org/wiki/GRIN2A	https://en.wikipedia.org/wiki/Glutamate_receptor	https://en.wikipedia.org/wiki/NMDA_receptor	human
4 4	T03	Team HS3	Tyeesha, Kamiah, Sherlyn	GRIN2B	Glutamate receptor ionotropic, NMDA 2B	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN2B_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN2B_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q13224	https://en.wikipedia.org/wiki/GRIN2B	https://en.wikipedia.org/wiki/Glutamate_receptor	https://en.wikipedia.org/wiki/NMDA_receptor	human
5 5	T04	Team HS4	Nia, Jocelyn, Arely	GRIN2C	Glutamate receptor ionotropic, NMDA 2C	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN2C_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN2C_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q14957	https://en.wikipedia.org/wiki/GRIN2C	https://en.wikipedia.org/wiki/Glutamate_receptor	https://en.wikipedia.org/wiki/NMDA_receptor	human
6 6	T05	Team HS5	Daniel, Laura	GRIN2D	Glutamate receptor ionotropic, NMDA 2D	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN2D_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN2D_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/O15399	https://en.wikipedia.org/wiki/GRIN2D	https://en.wikipedia.org/wiki/Glutamate_receptor	https://en.wikipedia.org/wiki/NMDA_receptor	human
7 7	T06	Team HS6	Romel, Israel	GRIN3A	Glutamate receptor ionotropic, NMDA 3A	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN3A_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN3A_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q8TCU5	https://en.wikipedia.org/wiki/GRIN3A	https://en.wikipedia.org/wiki/Glutamate_receptor	https://en.wikipedia.org/wiki/NMDA_receptor	human
8 8	T07	Team HS7	Thanh, Emanuel, Marvin	GRIN3B	Glutamate receptor ionotropic, NMDA 3B	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN3B_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-GRIN3B_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/O60391	https://en.wikipedia.org/wiki/GRIN3B	https://en.wikipedia.org/wiki/Glutamate_receptor	https://en.wikipedia.org/wiki/NMDA_receptor	human
9 9	T08	Team HS8	Zinna, Charlie, Josue	KCNA1	Potassium voltage-gated channel subfamily A member 1	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-KCNA1_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-KCNA1_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q09470	https://en.wikipedia.org/wiki/Kv1.1	https://en.wikipedia.org/wiki/Glutamate_receptor	https://en.wikipedia.org/wiki/NMDA_receptor	human
10 10	T09	Team HS9	Jan, Yuritzi, Nathaly	KCNK10	Potassium channel subfamily K member 10	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-KCNK10_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-KCNK10_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/P57789	https://en.wikipedia.org/wiki/KCNK10	https://en.wikipedia.org/wiki/Two-pore-domain_potassium_channel	NA	human
11 11	T10	Team HS10	Juan, Katia	KCNK2	Potassium channel subfamily K member 2	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-KCNK2_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-KCNK2_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/O95069	https://en.wikipedia.org/wiki/KCNK2	https://en.wikipedia.org/wiki/Two-pore-domain_potassium_channel	NA	human
12 12	T11	Team HS11	Loui, Nayeli, Q	Trpn1	Ion channel NompC	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-fish-Trpn1_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-fish-Trpn1_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q7T1G6	https://en.wikipedia.org/wiki/TRPN	https://en.wikipedia.org/wiki/Transient_receptor_potential_channel	http://www.trpchannel.org/	zebrafish
13 13	T12	Team HS12	Rosa, Noe	TRPV4	Transient receptor potential cation channel subfamily V member 4	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-TRPV4_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-TRPV4_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q9HBA0	https://en.wikipedia.org/wiki/TRPV4	https://en.wikipedia.org/wiki/Transient_receptor_potential_channel	http://www.trpchannel.org/	human
14 14	T14	Team MC2	ENKHZAYA BATTUGS, SHAINA CHARLES, VINESSA COFIELD	PIEZO1	Piezo-type mechanosensitive ion channel component 1	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-PIEZO1_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-PIEZO1_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q92508	https://en.wikipedia.org/wiki/PIEZO1	NA	NA	human
15 15	T15	Team MC3	JOAN DAVIS PINKNEY, PRECIOUS DEL ROSARIO, SAHAR FORMOLI	PIEZO2	Piezo-type mechanosensitive ion channel component 2	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-PIEZO2_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-PIEZO2_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q9H5I5	https://en.wikipedia.org/wiki/PIEZO2	NA	NA	human
16 16	T16	Team MC4	RUBEN GONZALEZ, JESSICA HUERTA, ASHLEY JONES	SCN5A	Sodium channel protein type 5 subunit alpha	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-SCN5A_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-SCN5A_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/Q14524	https://en.wikipedia.org/wiki/Nav1.5	https://en.wikipedia.org/wiki/Sodium_channel	NA	human
17 17	T17	Team MC5	DANTE MALONE, NICHOLLETTE MINIX, BIBIANA MORALES	SLC26A5	Prestin	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-SLC26A5_X_species-127-AGS-fasta.txt	https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-SLC26A5_X_species-127-AGS-treefile.txt	https://www.uniprot.org/uniprot/P58743	https://en.wikipedia.org/wiki/Prestin	https://en.wikipedia.org/wiki/Solute_carrier_family	NA	human

Do Sections A-F using TRPA1 or your Assigned Gene.

A: Build Alignment

Download TRPA1 and Assigned Gene Fasta files of protein sequences from giganticFISH. To do this:

1) Go to the TRPA1 fasta file that is available on giganticFISH: https://gigantic.fish/wp-content/uploads/2020/12/giganticFISH-human-TRPA1_X_species-127-AGS-fasta.txt

2) Use the Save Page option in your web browser to save the file to your computer - for example, in Chrome you would go: File > Save Page As > select the text option and save to your computer.

3) Go to your Assigned Gene Fasta file that is available on giganticFISH (see table above for link to your fasta file).

4) Use the Save Page option in your web browser to save the file to your computer - for example, in Chrome you would go File > Save Page As > select the text option and save to your computer.

Upload TRPA1 or your Assigned Gene fasta file to CIPRES - a supercomputer at UCSD and funded by the National Science Foundation (NSF) to provide dedicated support for large-scale phylogenetic analyses. Upload TRPA1 data on your first pass and your Assigned Gene data on your second pass through Sections A-F.
001000

To do this:

1) Create or select a folder to work in.

2) Create a label and 3) Save the folder

4) Select data folder - 5) Upload the Fasta file - 6) Provide a label - and 7) Save.

8) You should now have a Fasta file saved to a Project Data folder on CIPRES.

Run CIPRES MAFFT on the Fasta file to generate an alignment.

To do this:

1) Select the Tasks folder and 2) Create a new task.

2) Provide a label for the job - in this case you will be running MAFFT to generate a Multiple Sequence Alignment of the sequence data.

3) Select input data under the "Select Data" tab - for MAFFT alignment this would be your uploaded TRPA1 or Assigned Gene Fasta file of sequence data.

4) Select software to run under the "Select Tool" tab - for sequence alignment this would be the MAFFT software.

5) Select "MAFFT on XSEDE".

6) This will take you back to the Tasks Task Summary tab - now with MAFFT on XSEDE listed as Tool - no need to save things yet - we still have to check parameters (settings) for the software.

7) Adjust parameters by selecting the "Input Parameters" blue button or the "Set Parameters" tab - they do the same thing.

Maximum Hours --> 10

8) Click save parameter settings - and click OK for the popup box.

9) Click the Save Task button - generally avoid the "Save and Run Task" option, as it sometimes results in loss of all input information.

10) Run your new task - MAFFT alignment of the or Assigned Gene sequence data - using the Run Task button on the right. It will then change to View Status. After the job completes it will change to View Output.

CIPRES MAFFT alignment takes a few minutes, depending on the number of users on CIPRES at that time. TRPA1 Alignment takes just a few minutes - CIPRES will email you when it finishes.

B: View alignment

To give you a sense of what an alignment looks like you can download your MAFFT alignment and upload it to AlignmentViewer.

Go to AlignmentViewer

Upload your TRPA1 or Assigned Gene Alignment file you downloaded from CIPRES using the Choose File button on the right side of the AlignmentViewer webpage.

Explore your alignment noting regions of conservation vs divergence along the length of the alignment. Also note short sequences, as they may be incomplete and not fully assembled in the genome they came from - suggesting further work is needed in sequencing and assembling the genome.

You can save screen shots or other images that are of interest or that you have questions about and post on Slack and/or include in your presentation. This is beyond the required materials for the class but for those who are interested alignments are critical points of data evaluation in building trees and understanding gene evolution in protein engineering.

C: Build tree

Check that CIPRES MAFFT alignment completed successfully and save the file to your Data folder.

To do this:

1) Go to the Tasks folder. Mafft has finished its alignment if the Action button reads "View Output"

2) Click on the Action button once it changes from View Status to View Output. It often finishes prior to updating the Action button, so you can also click on the Action button when it reads View Status and check to see if there is output yet. Once it indicates output the job has completed.

3) Click on the Output View text (see above) to view the output.

4) Click on the output.mafft --> View text (above ) to view the alignment file.

5) Click on the Save To Current Folder button (see above) to save the alignment file to the Data folder.

7) Click the Save button to save the file. You will be taken back to the initial MAFFT output page.

8) Click on the Tasks folder to next prep a task to run CIPRES NCLConvertor.

Run CIPRES NCLConvertor on the TRPA1 or Assigned Gene Alignment file to produce a relaxed Phylip version of the alignment. The relaxed Phylip format is required for tree building. The general process to do this is similar to the steps followed above to run a MAFFT alignment, so we opted to leave out screen shots for NCLConvertor. Please see the examples above or ask if there is confusion as to how you should set things up and run things.

To do this:

1) Create a new Task.

2) Provide a Description - can name it NCLConvertor TRPA1 or Assigned Gene.

3) Select Input Data - selecting the output.mafft file that you saved to the Data folder.

4) Select Tool - selecting NCLConvertor.

5) Set the following parameters as follows:

Input Format --> Fasta

Maximum Hours to Run --> 1

Specify Data Type --> Protein

Output Format --> Relaxed Phylip

6) Click the Save Task button - and click OK for the popup box.

7) Click Run Task button on the right.

PAUSE
Until the job has completed: CIPRES NCLConvertor typically takes just a few minutes to complete.

8) Save the reformatted alignment file as above - the file you want to save is: out.aa.phy

Run CIPRES RAxML-NG on the Gene Tree Relaxed Phylip Alignment to produce a Gene Tree. The general process to do this is similar to the steps followed above to run a MAFFT alignment. To do this:

1) Create a new Task.

2) Provide a Description - can name it RAxML TRPA1 or Assigned Gene Alignment out.aa.phy file.

3) Select Input Data - selecting the out.aa.phy file that you saved to the Data folder.

4) Select Tool - selecting RAxML-NG.

5) Set the following parameters as follows:

Maximum Hours to Run --> 168 (or whatever max CIPRES now allows)

Sequence Type --> Protein

Number of Patterns --> 2000

6) Click the Save Task button - and click OK for the popup box.

7) Click Run Task button on the right.

PAUSE
Until the job has completed: CIPRES RAxML-NG TRPA1 or Assigned Gene takes several hours to run even days it seems like. CIPRES will email you when it finishes.

8) Save the file: infile.raxml.bestTree

D: View tree

Download the bestTree file from CIPRES.

Go to iTOL tree website.

Select the iTOL workspace (T01-T22) assigned to your team - see the spreadsheet above or ask if unsure what iTOL you are.

Click on the button Upload Tree File.

Rename the file to include your initials.

Click the Upload button.

Click on the Title name of the tree (blue text on right).

Click on one of the OGS (Outgroup Gene Set) sequences.

Select Tree Structure and then select Reroot the Tree Here in the pop-up.

Click on the Export tab in the Control panel.

Format --> PDF

Export Area --> Full Image

A pdf of the tree is automatically saved to your computer. We next want to annotate the tree for major groups of interest however iTOL is not very user-friendly, so we will do this in online software for annotating PDFs - Xodo.

E: Annotate tree

Annotate your Gene Tree - use the tree below (also on giganticFISH at https://gigantic.fish/wp-content/uploads/2020/11/species-tree-mini-skinny-anno.png )as a guideline as how to do this. For color in the box set opacity to 25%.

NOTE Jawless fishes = Agnathans and Bony fishes = Ray finned fishes

Here is an example of color settings in Xodo:

F: Post Tree

CRITICAL
Post your Annotated Gene Tree on the giganticFISH Slack Channel #TREES-PLEASE.

PAUSE
If you just finished TRPA1 data set you could stop here for now - or if there is time, you can start on your Assigned Gene. Also, feel free to continue and generate trees for additional genes, if you are interested in doing so. Make a new folder for each gene tree that you up build. This is important for keeping data and trees together and clear.

X: CONGRATULATIONS! YOU COMPLETED A GENE TREE :)

PAUSE
SCIENCE RESEARCH LEVELS 2 and 3 🙌 GENE TREE MILESTONE! 🙌

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giganticFISH Protocol: Channel Hunters - Gene Trees

Introduction

Materials

Procedure

Enzyme AatII

Enzyme lists

Cut sites visible on maps

Find enzyme

Clear selected

Show enzymes that cut

Show full history

In progress

Saved alignments

Name

Parent version

Folder

Topology

Start index

Locked

Creator

Created

Length

Export data

Welcome to Benchling!

Capture context, materials, and steps

Create protocol repositories for your lab

Insert tables with calculations

Attach protocols to Notebook entries

Create account

giganticFISH Protocol: Channel Hunters - Gene Trees

Introduction

Materials

Procedure

New digest

Saved digests

Enzyme AatII

Enzyme lists

Cut sites visible on maps

Find enzyme

Clear selected

Show enzymes that cut

Primers

Pairs

Version history

Show full history

Sequence Alignments

In progress

Saved alignments

Sequence Info

Tags

Name

Parent version

Folder

Topology

Start index

Locked

Creator

Created

Length

Export data

Enzyme Coloring