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phylogeny_protocol2 [2021/10/09 23:32] – 38.20.199.40phylogeny_protocol2 [2022/02/07 15:21] (current) – 134.190.232.106
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 +The GitHub resource for this protocol: https://github.com/zx0223winner/TreeTuner
 +
 **Background** **Background**
  
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   - **Treetrimmer** automatically reduces the number of leaves in a tree to few representatives for each user-defined operational taxonomical unit (OTU), like genus or species. However it is based on user-defined OTU.   - **Treetrimmer** automatically reduces the number of leaves in a tree to few representatives for each user-defined operational taxonomical unit (OTU), like genus or species. However it is based on user-defined OTU.
   - **Treemmer**, a simple tool based on an iterative algorithm to reduce size and evaluate redundancy of phylogenetic datasets. However, it is not related to hierarchical taxonomic terms, because he thought taxonomic categories are only a very rough proxy for genetic diversity. (The third one is what I added)   - **Treemmer**, a simple tool based on an iterative algorithm to reduce size and evaluate redundancy of phylogenetic datasets. However, it is not related to hierarchical taxonomic terms, because he thought taxonomic categories are only a very rough proxy for genetic diversity. (The third one is what I added)
-  - **SHOOT.bio** a single gene family tree. Shoot only offers a range of curated databases. Because they believe: the motivation for encouraging the use of curated databases over defaulting to the BLAST nr database is that these curated databases should make it easier to picture the evolution of the whole gene family rather than overwhelming the tree with thousands of near-identical hits.+  - **SHOOT.bio** a single gene family tree. Shoot only offers a range of curated databases. Because they believe: the motivation for encouraging the use of curated databases over defaulting to the BLAST nr database is that these curated databases should make it easier to picture the evolution of the whole gene family rather than overwhelming the tree with thousands of near-identical hits.(I added)
  
 So, I am thinking is there a better way to balance the methods/tools, I am not trying to re-programming and deny what they did. Actually, they did good job on some specific questions. An idea ,suddenly, coming into my mind, which might looks stupid, YES, I decided to name a new pipeline called TreeTuner which aims to combe some of the above methods and realize the coarse and fine-tuning of large phylogenetic datasets via reducing the redundancy and complexity. This might relief my goal on balancing the multiple available methods/tools a little bit. So, I am thinking is there a better way to balance the methods/tools, I am not trying to re-programming and deny what they did. Actually, they did good job on some specific questions. An idea ,suddenly, coming into my mind, which might looks stupid, YES, I decided to name a new pipeline called TreeTuner which aims to combe some of the above methods and realize the coarse and fine-tuning of large phylogenetic datasets via reducing the redundancy and complexity. This might relief my goal on balancing the multiple available methods/tools a little bit.
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 **Before you begin** **Before you begin**
  
-It was tricky than I thought even at the first step. Do I blast the data before the renaming the header or after? That does matter. If I blast before renaming the header, I need to pull out the BLAST hits sequence and rename them in a hierarchical taxonomic way. If I blast after the process of renaming the header, I need to reming all the database header in a hierarchical taxonomic way. Lost your mind? never mind! I will show you the difference here.+It was tricky than I thought even at the first step. Do I blast the data before renaming the header or after? That does matter. If I blast before renaming the header, I need to pull out the BLAST hits sequence and rename them in a hierarchical taxonomic way. If I blast after the process of renaming the header, I need to rename all the database header in a hierarchical taxonomic way. Lost your mind? never mind! I will show you the difference here.
  
 Here, We will deal with two databases: MMETSP and NCBI-nr. Why MMETSP? Because it included the marine microbial eukaryotic transcripts where NCBI-nr don't have all i.e., lots of algae, lots of Haptophytes, Rhodoophytes. It is also annoying for the different formats of headers. For example: Here, We will deal with two databases: MMETSP and NCBI-nr. Why MMETSP? Because it included the marine microbial eukaryotic transcripts where NCBI-nr don't have all i.e., lots of algae, lots of Haptophytes, Rhodoophytes. It is also annoying for the different formats of headers. For example:
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 </code> </code>
  
-This will be terrible long time doing that, 'cause I have not tested it. I also think it is not worth it unless you want to stick with one version to do intense studies in a period of time. The NCBI-nr version is updated quickly with time. So I prefer to download the latest version of NCBI-Nr, where they have the pre-compiled database files. Use can BLASTp against the database without using makeblastdb command. +This will be terrible long time doing that, 'cause I have not tested it. I also think it is not worth it unless you want to stick with one version to do intense studies in a period of time. The NCBI-nr version is updated quickly with time. So I prefer to download the latest version of NCBI-Nr, where they have the pre-compiled database files. User can BLASTp against the database without using makeblastdb command. 
  
 Let's say now you have finished the NCBI BLAST and MMETSP blast without renaming the database file at first. You will acquire a bunch of BLAST hits like this (by default, you are using tabular format output): Let's say now you have finished the NCBI BLAST and MMETSP blast without renaming the database file at first. You will acquire a bunch of BLAST hits like this (by default, you are using tabular format output):
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   * Step TWO: retrieve the Fasta sequence from the hits   * Step TWO: retrieve the Fasta sequence from the hits
  
-Why need to do this step, because without the hits sequence at hand, the MAFFT,BMGE,iqtree analysis is not available. As for MMETSP, you can use the python3 script: index_header_to_seq.py. I introduced in (http://129.173.88.134:81/dokuwiki/doku.php?id=phylogeny_protocol)+Why need to do this step, because without the hits sequence at hand, the downstream MAFFT,BMGE, Fasttree,iqtree analysis is not available. As for MMETSP, you can use the python3 script: index_header_to_seq.py. I introduced in (http://129.173.88.134:81/dokuwiki/doku.php?id=phylogeny_protocol)
  
 <code> <code>
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   - Please find the usage of acc2tax here: http://129.173.88.134:81/dokuwiki/doku.php?id=taxonomy_recovery   - Please find the usage of acc2tax here: http://129.173.88.134:81/dokuwiki/doku.php?id=taxonomy_recovery
   - acc2tax :Given a file of accessions or Genbank IDs (one per line), this program will return a taxonomy string for each. https://github.com/richardmleggett/acc2tax   - acc2tax :Given a file of accessions or Genbank IDs (one per line), this program will return a taxonomy string for each. https://github.com/richardmleggett/acc2tax
-  - /misc/db1/extra-data-sets/Acc2tax/Acc2tax_092021 (Up to date Sep 20, 2021) +  - The directory of the tool: /misc/db1/extra-data-sets/Acc2tax/Acc2tax_092021 (Up to date Sep 20, 2021) 
   - The taxa information is updated by NCBI weekly via https://ftp.ncbi.nih.gov/pub/taxonomy/; https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/   - The taxa information is updated by NCBI weekly via https://ftp.ncbi.nih.gov/pub/taxonomy/; https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/
  
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 </code> </code>
  
-Thanks for keeping reading until here, don't forget our goal is to acquire the header like this:+Note: If you only have hundreds of hits in a list, you can instead use the Taxonomy Common Tree - NCBI. Please read more from here: (http://129.173.88.134:81/dokuwiki/doku.php?id=phylogeny_protocol3)(https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi) 
 + 
 +Thanks for keeping reading until here, LOL don't forget our goal is to acquire the header like this:
  
 <code> <code>
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 So two python scripts(renaming_MMETSP.py and renaming_NCBI.py) are needed to proceed MMETSP and NCBI-nr,respectively, because they have very different naming format.  So two python scripts(renaming_MMETSP.py and renaming_NCBI.py) are needed to proceed MMETSP and NCBI-nr,respectively, because they have very different naming format. 
 +
 +Now I will introduce the usage of two Python scripts working on the naming issues. Let me clarify what you need to proceed this step again.
 +
 + * Input files for MMETSP:
 +    - A tabular file merged with all the genes' blast hits: merged_blast_mmetsp.txt
 +    - The fasta seq for all the hits: out_mmetsp.fasta
 +    - taxdump.tar.gz: https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/
 +
 +<code> 
 +# merged_blast_mmetsp.txt
 +ATCG00670.1 CP_0184350226_38269_Gloeochaete_wittrockiana 3.32e-76 54.922 98 193 292 196 1 193 98 289 N/A N/A
 +ATCG00670.1 CP_0184350226_38269_Gloeochaete_wittrockiana 3.32e-76 54.922 98 193 292 196 1 193 98 289 N/A N/A
 +ATCG00670.1 CP_0184656932_38269_Gloeochaete_witrockiana 4.26e-76 54.922 98 193
 +</code>
 +
 +<code>
 +# out_mmetsp.fasta
 +>CP_0113232432_97485_Prymnesium_parvum
 +XLRCLTRTPSLPSRLLATATPSRACPALSSALHRXASSAAFLRPSASASSCPSRCLSSTSRAPGASGSTQRAIPSXGGANGGWVNPLARPKGESLKKYGTDLNELARAGRLDPVIGRDEEIRRMVQVLSRRRKNNPVLIGEPGVGKTAIVEGLAQRIVDKEVPDSMRDARVIALDVGALVAGAKYRGEFE
 +</code>
 +
 +<code>
 +#taxdump.tar.gz
 +citations.dmp division.dmp gencode.dmp names.dmp readme.txt
 +delnodes.dmp gc.prt merged.dmp nodes.dmp
 +</code>
 +
 +Then run the python script renaming_MMETSP.py(link upcoming soon).
 +
 +<code>
 +python3 renaming_MMETSP.py
 +</code>
 +
 +The output fasta file will be like this:
 +
 +<code>
 +>Compsopogon_coeruleus@CP_0184679990_Eukaryota_Rhodophyta_Compsopogonophyceae_Compsopogonales_Compsopogonaceae_Compsopogon_Compsopogon_caeruleus_31354_Compsopogon_coeruleus
 +XNKTVGEKEKVDVGKKGGGGEEREMVGFVSDVFISLNLEWSRVGVGVVNSRGKRKVYAVGEFPGSSPGRTSVLVPQKEKVQKESKEKKRSHGGGKYKVLILNDAFNSMEYVAATLLRLIPGMTTELAWKVMKEAHENGAAVVGVWVFELAEAYCDAIQSAGIGSRIEPE
 +</code>
 +
 +
 + * Input files for NCBI:
 +   - A tabular file merged with all the genes' blast hits: merged_ncbi.txt
 +   - The fasta seq for all the hits: new.merged_ncbi.fasta
 +   - taxdump.tar.gz: https://ftp.ncbi.nlm.nih.gov/pub/taxonomy/
 +   - merged_taxon.txt
 +
 +Since NCBI has different header, so the merged_taxon.txt is needed. It was acquired via retrieve the all the hits' gene names from /misc/db1/extra-data-sets/Acc2tax/Acc2tax_092021/acc2tax_prot_all.txt file.
 +
 +<code>
 +# merged_taxon.txt
 +A1BI15 A1BI15.1 290317 166201813
 +A1WR17 A1WR17.1 391735 166214720
 +A4J7L9 A4J7L9.1 349161 172044337
 +A5D447 A5D447.1 370438 259585961
 +A6SY74 A6SY74.1 375286 226706516
 +A8F754 A8F754.1 416591 167008651
 +A8WPG6 A8WPG6.2 6238 300681014
 +</code>
 +
 +Then run the script (link upcoming):
 +<code>
 +python3 renaming_NCBI.py
 +</code>
 +
 +The output file will be look like this:
 +<code>
 +>Tarenaya_hassleriana@NCBI_XP_010551290.1_Eukaryota_Viridiplantae_Streptophyta_Streptophytina_Embryophyta_Tracheophyta_Euphyllophyta_Spermatophyta_Magnoliopsida_Mesangiospermae_eudicotyledons_Gunneridae_Pentapetalae_rosids_malvids_Brassicales_Cleomaceae_New_World_clade_Tarenaya_Tarenaya_hassleriana_28532
 +MESAICGRLALSPSTVFNSKPGEKHSLYKGPCGNHGFVMSLCASAVGKGGGLLDKPVIEKTTPGRESEFDLRKSRKMAPPYRVILHNDNFNRREYVVQVLMKVIPGMTLDNAVNIMQEAHHNGLAVVIICAQADAEEHCMQLRGNGLLSSIEPASGGGC
 +</code>
 +
 +Finally, (～￣▽￣)～ we have the desired BLAST hits headers from both MMETSP and NCBI-nr containing the hierarchical taxonomic terms. You can then merge the two files to play around what you are most familiar with seq aligning, seq trimming, and tree building. please read more http://129.173.88.134:81/dokuwiki/doku.php?id=phylogeny_protocol
 +
 +But after all of these, you will need the step to color your newick tree. Please find this script color_newick_tree.py via http://129.173.88.134:81/dokuwiki/doku.php?id=phylogeny_protocol4
  
 **2. Method Two: blasting after renaming the header** **2. Method Two: blasting after renaming the header**
  
-Still remember we mentioned earlier due to the different naming strategy, we have to decide to blast before renaming the header or after? Now I will introduce the usage of two Python scripts working on the naming issues.+Still remember we mentioned earlier due to the different naming strategy, we have to decide to blast before renaming the header or after? If you have read the Method One, you might feel it is from simple to complex. However, the Method TWO is from complex to simple, because it will take much longer than you thought to prepare the input files. But once you done, the rest of things could be much easier. Ok, let me stop eating around the corner. Our goal is to actually recreate the NCBI and MMETSP database headers like this:
  
-draw_color_tree.py+<code> 
 +>Compsopogon_coeruleus@CP_0184679990_Eukaryota_Rhodophyta_Compsopogonophyceae_Compsopogonales_Compsopogonaceae_Compsopogon_Compsopogon_caeruleus_31354_Compsopogon_coeruleus 
 +XNKTVGEKEKVDVGKKGGGGEEREMVGFVSDVFISLNLEWSRVGVGVVNSRGKRKVYAVGEFPGSSPGRTSVLVPQKEKVQKESKEKKRSHGGGKYKVLILNDAFNSMEYVAATLLRLIPGMTTELAWKVMKEAHENGAAVVGVWVFELAEAYCDAIQSAGIGSRIEPE 
 + 
 +>Tarenaya_hassleriana@NCBI_XP_010551290.1_Eukaryota_Viridiplantae_Streptophyta_Streptophytina_Embryophyta_Tracheophyta_Euphyllophyta_Spermatophyta_Magnoliopsida_Mesangiospermae_eudicotyledons_Gunneridae_Pentapetalae_rosids_malvids_Brassicales_Cleomaceae_New_World_clade_Tarenaya_Tarenaya_hassleriana_28532 
 +MESAICGRLALSPSTVFNSKPGEKHSLYKGPCGNHGFVMSLCASAVGKGGGLLDKPVIEKTTPGRESEFDLRKSRKMAPPYRVILHNDNFNRREYVVQVLMKVIPGMTLDNAVNIMQEAHHNGLAVVIICAQADAEEHCMQLRGNGLLSSIEPASGGGC 
 +</code> 
 + 
 +Then use makeblastdb command to make the database compiled files. Considering the size of NCBI ~100Gb and the MMETSP (~10GB), I have not really tested myself. But i assume it might take at least three days running. Here, I will simply provide the method for you to feel free to use. 
 + 
 +* As for MMETSP, the translated database header is like this:  
 + 
 +<code> 
 +>CP_0113232432_97485_Prymnesium_parvum 
 +</code> 
 + 
 +To pull out the taxa information, use the new python script rename_mmetsp_blastdb.py (links upcoming soon) 
 + 
 +<code> 
 +python3 rename_mmetsp_blastdb.py 
 +</code> 
 + 
 +Error1: 
 +<code> 
 +#Note: if not python v3, it will be error  
 +ImportError: No module named ete3 
 +</code> 
 + 
 +Error2: 
 +<code> 
 +from PyQt5 import QtGui, QtCore 
 +RuntimeError: the PyQt5.QtCore and PyQt4.QtCore modules both wrap the QObject class 
 +</code> 
 + 
 +To solve above error:use python3 
 +<code> 
 +source activate Unicycler-python3 
 +pip install six 
 +</code> 
 + 
 +Fist time running the script on MacOS, it might generate an error. (https://stackoverflow.com/questions/50236117/scraping-ssl-certificate-verify-failed-error-for-http-en-wikipedia-org) This will need you to allow the Macintosh HD > Applications > Python3.8 > double click on "Install Certificates.command" 
 + 
 +<code> 
 +####@TE809 ~ % /Applications/Python\ 3.9/Install\ Certificates.command ; exit; 
 + -- pip install --upgrade certifi 
 +Requirement already satisfied: certifi in /Library/Frameworks/Python.framework/Versions/3.9/lib/python3.9/site-packages (2021.10.8) 
 + -- removing any existing file or link 
 + -- creating symlink to certifi certificate bundle 
 + -- setting permissions 
 + -- update complete 
 +Saving session... 
 +...copying shared history... 
 +...saving history...truncating history files... 
 +...completed. 
 +</code> 
 + 
 +<code> 
 +#running 
 +/misc/scratch2/###/arabidopsis/CAM_MMETSP@perun3> python3 rename_mmetsp_blastdb.py  
 +NCBI database not present yet (first time used?) 
 +Updating taxdump.tar.gz from NCBI FTP site (via HTTP)... 
 +Done. Parsing... 
 +Loading node names... 
 +2369147 names loaded. 
 +253927 synonyms loaded. 
 +Loading nodes... 
 +</code> 
 + 
 +Then the latest taxdump.tar.gz will be downloaded via ETE3 package. The output file will be like this: 
 + 
 +<code> 
 +>Prymnesium_parvum@CP_0113232432_Eukaryota_Haptista_Haptophyta_Prymnesiophyceae_Prymnesiales_Prymnesiaceae_Prymnesium_Prymnesium_parvum_97485_Prymnesium_parvum 
 +XLRCLTRTPSLPSRLLATATPSRACPALSSALHRXASSAAFLRPSASASSCPSRCLSSTSRAPGASGSTQRAIPSXGGANGGWVNPLARPKGESLKKYGTDLNELARAGRLDPVIGRDEEIRRMVQVLSRRRKNNPVLIGEPGVGKTAIVEGLAQRIVDKEVPDSMRDARVIALDVGALVAGAKYRGEFEXRLKAVLADVSEAAGDVILFIDELHTVIGAGAADGAMDASNLLKPQLARGELSCVGATTLX 
 +>Prymnesium_parvum@CP_0113233658_Eukaryota_Haptista_Haptophyta_Prymnesiophyceae_Prymnesiales_Prymnesiaceae_Prymnesium_Prymnesium_parvum_97485_Prymnesium_parvum 
 +VASRXCEADDXAAAEGTRAVAMLPRLAIYLFAPLASASLVQLPQWPQRRLSPAGRLGLRPLPAAPRGSGQVQMVFDRFDRDAMRLVMDAQVEARKLGGSAVGTEHLLLAGTMQADAIQQALDRAGVKASGVRDAIRGPGGGSIPSLDGLFGLKAKDELLP 
 +</code> 
 + 
 +* As for NCBI-NR, the translated database header is like this: 
 + 
 +<code> 
 +>WP_048801694.1 ATP-dependent Clp protease ATP-binding subunit [Leuconostoc citreum]GEK62024.1 ATP-dependent Clp protease ATP-binding subunit ClpC [Leuconostoc citreum] 
 +MDNKYTSSAQNVLVLAQEQAKYFKHQAVGTEHLLLALAIEKEGIASKILGQFNVTDDDIREEIEHFTGYGM 
 +</code> 
 + 
 +<code> 
 +#So simply run  
 +python3 rename_ncbi_blastdb.py (link upcoming soon).  
 +#The input file will include: 
 +        fastaFile = '/db1/nr-nt-fasta-oct-2020/nr' 
 +        taxidFile = '/misc/db1/extra-data-sets/Acc2tax/Acc2tax_092021/acc2tax_prot_all.txt' 
 +</code> 
 + 
 +Then the desired output will be: 
 + 
 +<code> 
 +>Leuconostoc_citreum@NCBI_WP_048801694.1_Bacteria_Terrabacteria_group_Firmicutes_Bacilli_Lactobacillales_Lactobacillaceae_Leuconostoc_Leuconostoc_citreum_33964 
 +MDNKYTSSAQNVLVLAQEQAKYFKHQAVGTEHLLLALAIEKEGIASKILGQ 
 +</code> 
 + 
 +Directory to renamed MMETSP: /misc/scratch2/###/###/mmetsp 
 + 
 +Then with the two renamed database available, you could merge then by 'cat'. Then build the new merged database via 'makeblastdb'. Then Blast them again.8-) 
 + 
 +**3.Minimizing the redundancy and complexity of large phylogenetic datasets** 
 + 
 +Finally, after using two different methods, we can touch on the topic we raised up at very beginning. Coarse and fine-tuning large phylogenetic datasets via reducing the redundancy and complexity.  
 + 
 +1. **Coarse-tuning**: Let's start with the relatively simple one coarse-tuning via Treetrimmer (Maruyama et.al 2013) 
 + 
 +<code> 
 +ruby treetrimmer.rb sample/####_aligned_trimmed.newick sample/###_parameter_input.in sample/taxonomic_info.txt > ###_treetrimmer.newick 
 +</code> 
 + 
 +The "##..newick" and "###input.in" files can easily be prepared. The taxonomic_info.txt;however need to reformatted. 
 + 
 +<code> 
 +taxonomic_info.txt 
 +NP_563657 Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; malvids; Brassicales; Brassicaceae; Camelineae; Arabidopsis; Arabidopsis thaliana 
 +XP_002889406 Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; malvids; Brassicales; Brassicaceae; Camelineae; Arabidopsis; Arabidopsis lyrata; Arabidopsis lyrata subsp. lyrata 
 +</code> 
 + 
 +The taxonomic_info.txt can be created by acc2tax program. please read more from here:http://129.173.88.134:81/dokuwiki/doku.php?id=phylogeny_protocol3 
 + 
 +__Note: The acc2tax need the gene ID without version (e.g.NP_563657), so as the NCBI ID.__ Please find the usage of the program: http://129.173.88.134:81/dokuwiki/doku.php?id=taxonomy_recovery; http://129.173.88.134:81/dokuwiki/doku.php?id=phylogeny_protocol3 
 + 
 +<code> 
 +>WP_048801694.1 ATP-dependent Clp protease ATP-binding subunit [Leuconostoc citreum]GEK62024.1 ATP-dependent Clp protease ATP-binding subunit ClpC [Leuconostoc citreum] 
 +MDNKYTSSAQNVLVLAQEQAKYFKHQAVGTEHLLLALAIEKEGIASKILGQFNVTDDDIREEIEHFTGYGM 
 +</code> 
 + 
 +With the taxonomic_info.txt ready, you can get the tree file and another taxa file: 
 +<code> 
 +XP_026407875 Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; Ranunculales; Papaveraceae; Papaveroideae; Papaver; Papaver somniferum 2 4 
 +XP_034682772 Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliopsida; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; rosids; rosids incertae sedis; Vitales; Vitaceae; Viteae; Vitis; Vitis riparia 2 
 +</code> 
 + 
 +This tree give a rough tree diversity estimation. 
 + 
 + 
 +2. **Fine-tuning**  Laura Eme (2012-14) written in Perl 
 + 
 +<code> 
 + 
 +#!/bin/bash 
 +#$ -S /bin/bash 
 +. /etc/profile 
 +#$ -cwd 
 +#$ -o logfile 
 +#$ -pe threaded 20 
 +#export PATH=/scratch2/software/anaconda/bin:$PATH 
 + 
 +while read line 
 +do 
 + 
 +mafft --auto --thread 20 /misc/scratch2/####/$line.fasta >/misc/scratch2/####/aligned/$line.aligned.fasta 
 + 
 +/scratch2/software/anaconda/envs/bmge/bin/bmge -i /misc/scratch2/####/aligned/$line.aligned.fasta -t AA -m BLOSUM30 -of /misc/scratch2/xizhang/####/trimmed/$line.aligned.trimmed.fasta 
 + 
 +FastTree /misc/scratch2/####/trimmed/$line.aligned.trimmed.fasta > /misc/scratch2/####/fasttree/$line.aligned.trimmed.newick 
 + 
 +done <$1 
 +</code> 
 + 
 +let's say after the mafft,bmge,fasttree steps. You have the trimmed alignment and new wick tree. Now let's use the perl script to prune the leaves or trim the branches. 
 + 
 +<code> 
 +# These are files you will need. (links upcoming soon) 
 +# rm_inparal_rank.pl taxa_rank.txt 
 +# taxa_not_remove.txt trim2untrim.pl Instructions.txt lauralib.pm 
 + 
 +>perl rm_imparalogs <tree file> <alignment file> <distance cutoff> [taxa not to remove> <taxa rank> 
 +#Will remove sister sequences from the same rank. Will ignore taxa in the list "taxa not to remove". 
 +</code> 
 + 
 +It will yield the documents "###.removedSeq" and "###.fasttree". 
 + 
 +<code> 
 +> perl trim2untrim.pl [trimmed alignement] [untrimmed alignment] 
 +#Will remove sequences from the untrimmed alignement based on sequences present in the trimmed alignement 
 +</code>
  
-**3. Step-by-step Protocol**+Based on the trimmed aligned seq, you can re-analysis more rigorous downstream IQ-tree analysis.
  
-**4. Limitation**+Note: not all genes' species have taxa.This have nothing to do with the updates of NCBI taxonomy.
  
 +The '0' in Gene name 'CP_0177652116_0_Stygamoeba_regulata_BSH-02190019' is not a NCBI taxid. 
  
  
-<Last updated by Xi Zhang on Oct 6th,2021> upcoming+<Last updated by Xi Zhang on Oct 6th,2021>
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