min viable analysis

Lon.Groel.Ttest.csv

@@ -0,0 +1,16 @@
+protein ,edge,edge length,species,EL/ EL of outgroup,phylogeny,A,B
+Lon,1,1.0521435,Mycobacterium 6IHG A,0.767797968,A,0.767797968,5.82E-06
+,2,1.0521435,Thermus 4EIW A,0.767797968,A,0.767797968,5.82E-06
+,3,0.3181955,MRCA of 1 &2,0.232202032,A,0.232202032,5.83E-06
+,4,1.370339,Thermus 1IXS B,1,A,1,5.83E-06
+,5,0,MRCA of 4 & 6,0,A,0,8.35E-01
+,6,1.370339,Escherichia 6N2I A,1,A,1,8.35E-01
+,,,,,,,1.00E+00
+GroEL,1,2.00E-06,Thermus 1WE3 A,5.82E-06,B,,1.65E-01
+,2,2.00E-06,Thermus 1SRV A,5.82E-06,B,,
+,3,2.00E-06,Mycobacterium 3RTK A,5.83E-06,B,,
+,4,2.00E-06,Mycobacterium 1 SJP A,5.83E-06,B,,
+,5,2.86E-01,MRCA of 4 & 5,8.35E-01,B,,
+,6,2.86E-01,MRCA of 1 & 2,8.35E-01,B,,
+,7,3.43E-01,Escherichia K-12 5OPX A,1.00E+00,B,,
+,8,5.67E-02,MRCA of 7 & all,1.65E-01,B,,

README.md

@@ -1,50 +1,73 @@
 # Phylogenetic Biology - Final Project
 
-## Guidelines - you can delete this section before submission
+# Comparing Evolution Rates of Protein Quality Control Genes
 
-This repository is a stub for your final project. Fork it, develop your project, and submit it as a pull request. Edit/ delete the text in this readme as needed.
+## Introduction and Goals
 
-Some guidelines and tips:
+The goal of my project is to answer the question, "Do proteases and chaperones evolve at a similar rate?"
+This question is prompted by a recent study available on bioRxiv on proteome expansion across the tree of life (Rebeaud et al., 2020). They write extensively about the evolution of chaperones and co-chaperones, but say little about proteases. However, it has been well-catalogued that chaperones and proteases play different (but arguably equally important) roles in maintaining proteostasis in a cell (Gottesman et al., 1997). Furthermore, the network of chaperones and proteases that make up protein quality control (PQC) networks are necessary for cell tolerance of perturbations, and, acting as a mechanism of robustness are thus a driving force of evolution. 
 
-- Use the stubs below to write up your final project. Alternatively, if you would like the writeup to be an executable document (with [knitr](http://yihui.name/knitr/), [jupytr](http://jupyter.org/), or other tools), you can create it as a separate file and put a link to it here in the readme.
+The methods I will use to do this are...
+I will start by gathering protein sequence data on NCBI. I will find a region within Lon protease that has a high consensus with other microbes. 
+Then, I will gather protein sequence data on NCBI for a chaperon, GroEL.
 
-- For information on formatting text files with markdown, see https://guides.github.com/features/mastering-markdown/ . You can use markdown to include images in this document by linking to files in the repository, eg `![GitHub Logo](/images/logo.png)`.
+For the minimum viable analysis, I will use the protein sequences for Lon protease and GroEL chaperone to run BLAST and search for homologous regions in other proteases/chaperones in different bacteria. 
+I will upload this data to the Yale computing cluster (Grace), then run IQ-tree to make a phylogenetic tree of the homologous proteases from different bacteria. 
 
-- The project must be entirely reproducible. In addition to the results, the repository must include all the data (or links to data) and code needed to reproduce the results.
+Thus, each edge will represent a specific (homologous) protein region from different bacteria. I will also use R to estimate and compare evolutionary rates of time based on the phylogenies I create. 
 
-- If you are working with unpublished data that you would prefer not to publicly share at this time, please contact me to discuss options. In most cases, the data can be anonymized in a way that putting them in a public repo does not compromise your other goals.
+I will be limited by what is publicly available, but I hope to implement the methods I learn in this project on my own sequence data as my thesis progresses. This will ultimately be sequence data from nearshore bacterial communities in the Long Island Sound. 
 
-- Paste references (including urls) into the reference section, and cite them with the general format (Smith at al. 2003).
+## Methods
 
-- Commit and push often as you work.
+First, I searched for Lon protease in E coli on NCBI. I selected AAC36871.1 for my query ID and BLASTed it (BLASTP) using the protein database (PDB) for all prokaryotes (taxid 2). 
 
-OK, here we go.
+I selected nine of the most diverse species available from the results and chose the sequences among those species that had the highest coverage from the query.
+I downloaded the aligned file in fasta format and updated the header to label Genus_species_SequenceID (lon.renamed.fasta). 
+I then uploaded this file to the cluster, as well as two job scripts I had made using MAFFT for alignment and IQtree and analyzation. 
 
-# Title of my project
+I ran my job scripts, creating an aligned file, then ran IQTREE. 
 
-## Introduction and Goals
+I created an rmd file and made a phylogenetic tree of my species based on the Lon protease sequences.
 
-The goal of my project is to answer the question, What is...?
+THINGS THAT I NEED TO FIX and NEXT STEPS:
+My phylogeny doesn't really make sense because I have two E. coli sequences that are mapped sooooo distantly from each other (moreso than everything else). I'm guessing this is an error with a sequence I chose from NCBI (maybe one of them was not truly an E. coli protease sequence, but a sequence from another species that had been transformed into E. coli? I need to take a closer look at these sequences and sequence info on ncbi)
 
-The methods I will use to do this are...
+Next, I searched for a GroEL chaperonin on NCBI. I selected WP_000729117.1 for my query ID and BLASTed it (BLASTP) under the same filters as Lon protease: pdb, prokaryotes. 
 
-The data I will use are (my own data/ data publicly available at YYY/ simulations)
+I also added "filters". My filters were the genus and species names of all bacteria whose sequences I used in my Lon protease analysis. My results yielded sequences for E. coli, Mycobacterium tuberculosis, and Thermus thermophilus. The other species were not found. 
 
-## Methods
+I have six total protein sequences for GroEL, which I aligned and analyzed using MAFFT and IQTREE, with the same job scripts as for Lon protease. 
+
+The models used for the analyses of Lon and GroEL were LG + GR and LG + F + G4, respectively.
+
+Next I created two small phylogenetic trees of each of these in Rstudio. I rooted my trees with E. coli and checked with the is.root() function. This is important so that I can make accurate statements about the divergence rates of chaperones and proteases. I then made each tree ultrametric. 
+
+I calculated the edgelengths using phytools, then made an excel file to normalize each edge length to the edge length of E. coli. 
+
+Then I completed F and T tests (see results). 
 
-The tools I used were... See analysis files at (links to analysis files).
 
 ## Results
 
-The tree in Figure 1...
+The tree in Figure 1 is Lon protease and the tree in Figure 2 is GroEL chaperone. I did an F test to look for significant difference in edge length within each tree. This measured the variance in evolution rate of Lon across species. It also measured the variance in evolution rate of GroEL across species. The p-value was 0.8953, meaning there was no significant difference in rate evolution across species within each tree. 
+
+Then I did a T test to look for significant diferences in edge lengths between the two trees (Lon and protease). This answers the overall question of my project. The p-value was 0.264, not significant. 
 
 ## Discussion
 
-These results indicate...
+These results indicate that evolution rate between Lon and GroEL was not statistically different. 
 
-The biggest difficulty in implementing these analyses was...
+The biggest difficulty in implementing these analyses was figuring out how to normalize the edge lengths and make sure I was appropriately comparing edge lengths between two different trees (I'm sure there is a better way than I did). 
 
-If I did these analyses again, I would...
+If I did these analyses again, I would include way more bacteria with an outgroup in eukaryota or archaea. I would also couple another characteristic of my bacteria to these proteins. 
+
+*** I did another analysis in which I added lots more taxa to my lon protease phylogeny, including a total of 25 tips and 48 total edges. This helped resolve some of the discrepancies in my phylogeny, although there were still some random E. coli lineages still all over the place. I'm including some of the extra files here. In the future I intend to do further analyses to test the variance between genuses within a phylogeny to see how adding taxa will affect my results. I expect that they will further support the same conclusions that chaperones and proteases have similar evolution rates.
 
 ## References
 
+https://www.biorxiv.org/content/10.1101/2020.06.08.140319v2
+
+http://genesdev.cshlp.org/content/11/7/815.full.pdf
+
+https://academic.oup.com/mbe/article/25/7/1307/1041491

groel.aligned.fasta

@@ -0,0 +1,66 @@
+>Escherichia_coli_4PKN_A
+MAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREI
+ELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVAAGMNPMDLKRGI
+DKAVTAAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEAMDKVGKEGVITVEDG
+TGLQDELDVVEGMQFDRGYLSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAV
+AKAGKPLLIIAEDVEGEALATLVVNTMRGIVKVAAVKAPGFGDRRKAMLQDIATLTGGTV
+ISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIEEATSDY
+DREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVALI
+RVASKLADLRGQNE-DQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGN--YG
+YNAATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKNDAADLGAAGG
+MGGMGGMGGMM
+>Escherichia_Coli_K-12_5OPX_A
+MAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREI
+ELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVACGMNPMDLKRGI
+DKAVTAAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEAMDKVGKEGVITVEDG
+TGLQDELDVVEGMQFDRGYLSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAV
+AKAGKPLLIIAEDVEGEALATLVVNTMRGIVKVAAVKAPGFGDRRKAMLQDIATLTGGTV
+ISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIEEATSDY
+DREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVALI
+RVASKLADLRGQNE-DQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGN--YG
+YNAATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKNDAADLGAAGG
+MGGMGGMGGMM
+>Mycobacterium_tuberculosis_3RTK_A
+--AKTIAYDEEARRGLERGLNALADAVKVTLGPKGRNVVLEKKWGAPTITNDGVSIAKEI
+ELEDPYEKIGAELVKEVAKKTDDVAGDGTTTATVLAQALVREGLRNVAAGANPLGLKRGI
+EKAVEKVTETLLKGAKEVETKEQIAATAAISAG-DQSIGDLIAEAMDKVGNEGVITVEES
+NTFGLQLELTEGMRFDKGYISGYFVTDPERQEAVLEDPYILLVSSKVSTVKDLLPLLEKV
+IGAGKPLLIIAEDVEGEALSTLVVNKIRGTFKSVAVKAPGFGDRRKAMLQDMAILTGGQV
+ISEEVGLTLENADLSLLGKARKVVVTKDETTIVEGAGDTDAIAGRVAQIRQEIENSDSDY
+DREKLQERLAKLAGGVAVIKAGAATEVELKERKHRIEDAVRNAKAAVEEGIVAGGGVTLL
+QAAPTLDEL--KLEGDEATGANIVKVALEAPLKQIAFNSGLEPGVVAEKVRNLPAG--HG
+LNAQTGVYEDLLAAGVADPVKVTRSALQNAASIAGLFLTTEAVVADKPEKEKASV-----
+-----------
+>Mycobacterium_tuberculosis_1SJP_A
+-------------------------------------------WGAPTITNDGVSIAKEI
+ELEDPYEKIGAELVKEVAKKTDDVAGDGTTTATVLAQALVREGLRNVAAGANPLGLKRGI
+EKAVEKVTETLLKGAKEVETKEQIAATAAISAG-DQSIGDLIAEAMDKVGNEGVITVEES
+NTFGLQLELTEGMRFDKGYISGYFVTDPERQEAVLEDPYILLVSSKVSTVKDLLPLLEKV
+IGAGKPLLIIAEDVEGEALSTLVVNKIRGTFKSVAVKAPGFGDRRKAMLQDMAILTGGQV
+ISEEVGLTLENADLSLLGKARKVVVTKDETTIVEGAGDTDAIAGRVAQIRQEIENSDSDY
+DREKLQERLAKLAGGVAVIKAGAATEVELKERKHRIEDAVRNAKAAVEEGIVAGGGVTLL
+QAAPTLDEL--KLEGDEATGANIVKVALEAPLKQIAFNSGLEPGVVAEKVRNLPAG--HG
+LNAQTGVYEDLLAAGVADPVKVTRSALQNAASIAGLFLTTEAVVADKPEKEKASV-----
+-----------
+>Thermus_thermophilus_1WE3_A
+--AKILVFDEAARRALERGVNAVANAVKVTLGPRGRNVVLEKKFGSPTITKDGVTVAKEV
+ELEDHLENIGAQLLKEVASKTNDVAGDGTTTATVLAQAIVREGLKNVAAGANPLALKRGI
+EKAVEAAVEKIKALAIPVEDRKAIEEVATISAN-DPEVGKLIADAMEKVGKEGIITVEES
+KSLETELKFVEGYQFDKGYISPYFVTNPETMEAVLEDAFILIVEKKVSNVRELLPILEQV
+AQTGKPLLIIAEDVEGEALATLVVNKLRGTLSVAAVKAPGFGDRRKEMLKDIAAVTGGTV
+ISEELGFKLENATLSMLGRAERVRITKDETTIVGGKGKKEDIEARINGIKKELETTDSEY
+AREKLQERLAKLAGGVAVIRVGAATETELKEKKHRFEDALNATRAAVEEGIVPGGGVTLL
+RAISAVEELIKKLEGDEATGAKIVRRALEEPARQIAENAGYEGSVIVQQILAETKNPRYG
+FNAATGEFVDMVEAGIVDPAKVTRSALQNAASIGALILTTEAVVAEKPEKKESTPASAG-
+-----------
+>Thermus_thermophilus_1SRV_A
+------------------------------------------------------------
+------------------------------------------------------------
+------------------------------------------------------------
+-----------GYQFDKGYISPYFVTNPETMEAVLEDAFILIVEKKVSNVRELLPILEQV
+AQTGKPLLIIAEDVEGEALATLVVNKLRGTLSVAAVKAPGFGDRRKEMLKDIAAVTGGTV
+ISEELGFKLENATLSMLGRAERVRITKDETTIVGG-------------------------
+------------------------------------------------------------
+------------------------------------------------------------
+------------------------------------------------------------
+-----------

groel.aligned.fasta.bionj

@@ -0,0 +1 @@
+(((Mycobacterium_tuberculosis_1SJP_A:0.00385615,Mycobacterium_tuberculosis_3RTK_A:-0.00385515):0.35672656,(Escherichia_Coli_K-12_5OPX_A:0.00265439,Escherichia_coli_4PKN_A:-0.00085149):0.38389650):0.16773503,Thermus_thermophilus_1WE3_A:0.06670723,Thermus_thermophilus_1SRV_A:-0.06670623);

groel.aligned.fasta.ckp

@@ -0,0 +1,28 @@
+--- # IQ-TREE Checkpoint ver >= 1.6
+CandidateSet:
+ 00: -2951.04224672 (0:0.0000021461,1:0.0018133378,((2:0.0000018572,3:0.0000021461):0.3394733528,(4:0.0000018452,5:0.0000021461):0.233264319):0.3980136494);
+ 01: -2985.15046931 (0:0.0000027731,1:0.0018179788,(((2:0.0000025611,3:0.0000027731):0.4377040088,5:0.0000020056):2.797999623e-06,4:0.1127298559):0.4954904574);
+ 02: -2985.15259999 (0:0.0000027731,1:0.0018166105,(((2:0.0000025611,3:0.0000027731):0.4345531537,4:0.1132542117):2.797999623e-06,5:0.0000020056):0.4944965454);
+ 03: -3045.54982119 (0:0.0000027731,1:0.0018355072,(2:0.0000020405,(3:0.0000028589,(4:0.0000021374,5:0.0000027731):0.5943542041):2.344671727e-06):0.7656996935);
+ 04: -3045.55009094 (0:0.0000027731,1:0.0018355470,((2:0.0000020405,(4:0.0000021374,5:0.0000027731):0.5944511323):2.914313782e-06,3:0.0000028589):0.7657230949);
+ 05: -3063.99972634 (0:0.0000026805,(1:0.0016281397,(4:0.0000022529,5:0.0000027731):0.6655388849):2.773064278e-06,(2:0.0000026858,3:0.0000027731):0.7644423912);
+ 06: -3064.13421067 (0:0.0000028667,(1:0.0015781695,(2:0.0000026858,3:0.0000027731):0.7279690918):2.773064278e-06,(4:0.0000022529,5:0.0000027731):0.6519891353);
+PhyloTree:
+ newick: (0:0.0000021461,1:0.0018133378,((2:0.0000018572,3:0.0000021461):0.3394733528,(4:0.0000018452,5:0.0000021461):0.233264319):0.3980136494);
+RateGamma:
+ gamma_shape: 1.200230213
+StopRule:
+ curIteration: 101
+ start_real_time: 1604352861
+ time_vec: 
+boot_consense_logl: 0
+contree_rfdist: -1
+finished: true
+finishedCandidateSet: true
+finishedModelFinal: true
+finishedModelInit: true
+iqtree:
+ command:  -s groel.aligned.fasta -nt AUTO
+ seed: 132281
+ start_time: 1604352758
+ version: 1.6.12