The aim of this exercise is to get the phylogenetic relationships of the ortholog sequences found previously. Therefore we choose an ortholog group and expand it even further with orthologs from InParanoid and orthoMcl. We align the sequences of the proteins and perform a maximum-likelyhood-tree-reconstruction. Additional a strict-consensus tree is created from 100 boot-strap trees.
-
From the HaMStR-output select those genes that identified more than 2 Octopus ortholog
Following shell script is used to extract the genes: #!/bin/bash for f in $(ls [0-9]*.fa) do octo_count=$(grep "|comp" $f | wc -l) if [ "$octo_count" -gt "2" ] then echo $f egrep -h -o "\|[[:alnum:]_]+\|[[:alnum:]_]+" $f fi done -
List the ids of these gene
111219.fa |caeel_3|5516 |schma_370|4728 |apime_353|18256 |capsp_2853|8033 |dappu_1391|23169 |helro_2711|11899 |lotgi_2713|16857 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111358.fa |caeel_3|10476 |schma_370|4731 |apime_353|5024 |capsp_2853|21599 |dappu_1391|19556 |helro_2711|13303 |lotgi_2713|2943 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111369.fa |caeel_3|14598 |schma_370|3947 |apime_353|17677 |capsp_2853|22830 |dappu_1391|7337 |helro_2711|5863 |lotgi_2713|5776 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111520.fa |caeel_3|11517 |schma_370|8248 |apime_353|23146 |capsp_2853|7425 |dappu_1391|22671 |helro_2711|17716 |lotgi_2713|5492 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111609.fa |caeel_3|9926 |schma_370|13077 |apime_353|2427 |capsp_2853|23241 |dappu_1391|17979 |helro_2711|21194 |lotgi_2713|23345 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111677.fa |caeel_3|22624 |schma_370|8211 |apime_353|12837 |capsp_2853|19344 |dappu_1391|9449 |helro_2711|237 |lotgi_2713|13141 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111749.fa |caeel_3|6030 |schma_370|9776 |apime_353|6067 |capsp_2853|24106 |dappu_1391|28864 |helro_2711|22555 |lotgi_2713|7727 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111761.fa |caeel_3|9663 |schma_370|11539 |apime_353|22268 |capsp_2853|3637 |dappu_1391|10138 |helro_2711|22489 |lotgi_2713|680 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111794.fa |caeel_3|23833 |schma_370|5061 |apime_353|11132 |capsp_2853|3356 |dappu_1391|10142 |helro_2711|17724 |lotgi_2713|965 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111813.fa |caeel_3|26134 |schma_370|7819 |apime_353|7773 |capsp_2853|12100 |dappu_1391|3778 |helro_2711|15086 |lotgi_2713|8606 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111846.fa |caeel_3|14543 |schma_370|5036 |apime_353|13575 |capsp_2853|11202 |dappu_1391|23865 |helro_2711|12434 |lotgi_2713|22445 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111923.fa |caeel_3|13364 |schma_370|8512 |apime_353|3053 |capsp_2853|19375 |dappu_1391|3486 |helro_2711|20870 |lotgi_2713|2343 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111934.fa |caeel_3|18303 |schma_370|1216 |apime_353|26041 |capsp_2853|27103 |dappu_1391|12579 |helro_2711|12423 |lotgi_2713|11544 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 111949.fa |caeel_3|12561 |schma_370|3662 |apime_353|6810 |capsp_2853|11911 |dappu_1391|9301 |helro_2711|23250 |lotgi_2713|10031 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 112153.fa |caeel_3|17302 |schma_370|1096 |apime_353|18864 |capsp_2853|27850 |dappu_1391|19307 |helro_2711|11045 |lotgi_2713|22808 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 112257.fa |caeel_3|4924 |schma_370|851 |apime_353|15550 |capsp_2853|12362 |dappu_1391|14248 |helro_2711|14564 |lotgi_2713|19584 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 112258.fa |caeel_3|26012 |schma_370|7431 |apime_353|5096 |capsp_2853|16707 |dappu_1391|7511 |helro_2711|1827 |lotgi_2713|16440 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea 112347.fa |caeel_3|11878 |schma_370|6811 |apime_353|25501 |capsp_2853|9352 |dappu_1391|8206 |helro_2711|7187 |lotgi_2713|17065 |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea |lotgi_2713|lottia_gigantea The various repetitions of the gene "lotgi_2713|lottia_gigantea" are found orthologs with our sample (c0-c10) -
Choose one of these genes. Expand the ortholog cluster
cat 111923.fa | grep -A 1 "|caeel_3|13364" >111219|lotgi_2713|16857 MIGRKEKERERPYYRDPDDPEYIKDLQRPAVIKEDLSEMERRKRVQQILESKSFCHELEEVIRQECDTARTDPDHLQVLQKLSDLTVPQGNMSFGNLHTYGGNTIAIADLRGNEKYSKAERIQRNKLACLFRLADLFQWSQGIHNEISYRTNDEDNTFLMNPFGLLYHEITAATIVKIDENGKILDCGTLKAGVNQPAFLLHSAIYKAHPMVRCILHMHTAIVAAVASMKCGLLPLCKEAMVLGPVGYHDYQDIGDDDIQFDEIIANLGDKNVLFLRNQGFLVVGDTIEHATFLANNTVIACETQVRAARAGLDNLIIPEEKAIQRAFRNSRNTNSLKRNGTVDWRVGELEWESWMRVLDHANFQTGHVYRQPQLRPKSAMSTSMVNNNDVAVPPTTSAYGQIDETNLESVSAHRLALLRKEQERVRWMNSPNAYQKVEFLEYGADNPKKITKWVHDVNVPSASGTPVKISSVHQFSPASSNPKEFKEKQKAIKENNRLGTLSAGPQSQILDSVTYEDIALLIKPDNDGTVGQSSTADRAILIGTASKGIIDRQFQHHAQVYHQIYAPNPFSVETDADIKKYVDMVKAKNSQSAPVSARSGYSQYDEVEADTVSLMQGVREHKLSQAALSASDDGLNAGISPNNVRTSEESVNTSYMSQSVVFDCDHPSPMSISTEYPEKVKMTRFSSTQGTSEGNTTSRSCTTASEEEKPTKDEKKKKKKGFLSFMRKKDKa. with sequences from InParanoid. (There is no need to use all orthologs but make sure that you maintain the diversity within the taxon set).
Expanding the cluster by: Anopheles gambiae AGAP004852-PA gi|158293044 Drosophila ananassae FBpp0115800 gi|194753162 Brugia malayi XP_001902377.1 tr|A8QG43 + Mus musculus ENSMUSP00000109979 tr|Q8BJT2 Acyrthosiphon pisum XP_001948434.1 gi|193615505 \* Homo Sapiens ENSP00000264758 gi|29826321 Bos taurus ENSBTAP00000011966 gi|149642577 \* Danio rerio ENSDARP00000093507 gi|121582316b. with sequences from orthoMCL. Which orthoMCL cluster do you identify and how many species and sequences are represented? Again, there is no need to use all orthologs, make a reasonable selection of 10 to 15 species/sequences that should cover the full phylogenetic range in the ortholog set
Cluster OG5_127052 with 149 sequences from 83 species. Drosophila melanogaster dmel|FBpp0085732 Emericella nidulans anid|AN5007 Equus caballus ecab|XP_001497126 ecab|XP_001497126 Gallus gallus ggal|ENSGALP00000013902 \* Homo sapiens hsap|ENSP00000264758 Macaca mulatta mmul|XP_002799318 + Mus musculus mmus|ENSMUSP00000032069 Schistosoma mansoni sman|Smp_083040.2 Takifugu rubripes trub|ENSTRUP00000027790 \* Danio rerio drer|ENSDARP00000093507 \* also used from inparaniod + ortholog used from inparanoidc. What do you learn about the likely function of your gene?
-
Align the two sequence files with mafft choosing the most sensitive parameter options. List these options.
From the man-page of mafft: *G-INS-i (suitable for sequences of similar lengths; recommended for <200 sequences; iterative refinement method incorporating global pairwise alignment information): mafft --globalpair --maxiterate 1000 input [> output] ginsi input [> output] mafft --globalpair --maxiterate 1000 orthomcl.fa > orthomcl_mafft mafft --globalpair --maxiterate 1000 inparanoid.fa > inparanoid_mafft -
Convert the alignments into PHYLIP format using clustalw -convert and compute an ML and a distance tree each for both alignments. Perform the tree reconstructions via a web server of your choice. We suggest http://www.atgc-montpellier.fr/phyml. Perform for each analysis 100 bootstrap replicates.
Used commands to convert the files: clustalw -convert -infile=orthomcl_mafft -outfile=orthomcl_phyl -output=PHYLIP clustalw -convert -infile=inparanoid_mafft -outfile=inparanoid_phyl -output=PHYLIP ML-trees obtained by the website suggested: Inpranoid maximum likely tree:
OrthoMcl maximum likely tree:
a. Infer the consensus tree from the 100 bootstrap replicates and draw it with figtree.
First we used figtree to export the 100 bootstrap trees to the nexus format. Next we used paup to compute the strict consensus tree:
Starting paup: `paup inpara.nexus`
In paup: `contree all/strict=yes treefile=inpara_strict_consensus`
paup contree all/strict=yes treefile=inpara_strict_consensus
Inparanoid strict consensus tree:
OrthoMcl strict consensus tree:
b. Locate the likely root of the tree.
c. What can you infer about the relationships of the three putative co-orthologs from the tree?
The co-orthologs are too many and too close on the tree, as to assume, that every single one is a single ortholog. It's much more likely, that sequencing-errors and/or different transcripts of the same gene led to 10 different orthologs. The tree suggests, that there are 4 groups: seq2+seq6, seq5+seq3+seq1+seq9, seq4+seq10, seq8+seq7 and thus 4 paralogs inside this species.
With the used tools we managed to put the found orthologs in some perspective to other species - the closest related species is the lottia gigantea. Also we found, that of the analysed gene we probably had different transcripts and/or sequencing errors, as all the genes where much to close on the tree.