GeneInvestigator (GI)

An application for user-friendly state of the art molecular sequence analysis.

Software availability

Github repository https://github.com/aglucaci/GeneInvestigator

Retreive input data from NCBI Orthologs

Transcript and Protein data from orthologous sequences.

For example, if we are interested in the TP53 gene: https://www.ncbi.nlm.nih.gov/gene/7157/ortholog/?scope=117570&term=TP53

Download all information:

• Tabular data (Contains accession numbers, species scientific name, and other metadata)
• Reference transcript sequence.
• Reference protein sequence.

Typically performed on one gene per species, but all transcripts per species can also be analyzed as an option.

Pipeline

Introduction for TP53

Excerpt from: https://www.ncbi.nlm.nih.gov/gene/7157

This gene encodes a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. The encoded protein responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. Mutations in this gene are associated with a variety of human cancers, including hereditary cancers such as Li-Fraumeni syndrome. Alternative splicing of this gene and the use of alternate promoters result in multiple transcript variants and isoforms. Additional isoforms have also been shown to result from the use of alternate translation initiation codons from identical transcript variants (PMIDs: 12032546, 20937277). [provided by RefSeq, Dec 2016]

Example Results

Quick summary text

{Todo}

Number of species in alignment
Number of sites in alignment
GC content?
Summarize positive and negative sites

Results of BUSTEDS analysis for a gene-level overview

Figure 1. BUSTEDS found evidence found evidence (LRT, p-value ≤ 0.05) of gene-wide episodic diversifying selection in the selected test branches of your phylogeny.

Describe differences between omega {1,2,3} value and proportion between unconstrained and constrained model. Anything to say with ER plots? Threshold the ER plot?

Results of MEME analysis for episodic positive selection

Plotting everything but highlighting (via color) the significant positive sites.

Figure 2. MEME analysis of your gene of interest found 126 of 1167 sites to be statisically significant (p-value <= 0.1)

Results of FEL analysis for negative selection

Only plotting the negative sites here

Figure 3. FEL analysis of your gene of interest found 411 of 1167 sites to be statisically significant (p-value <= 0.1) for pervasive negative/purifying selection

Results of FUBAR analysis for episodic selection

Plot everything, but highlight the negative sites

"ProbNegative" here corresponds to "Prob[alpha>beta]" from the FUBAR output.

Figure 4. FUBAR analysis of your gene of interest found 418 of 1167 sites to be statisically significant (posterior probability threshold 0.9) for episodic negative/purifying selection. FUBAR analysis of your gene of interest found 2 of 1167 sites to be statisically significant (posterior probability threshold 0.9) for episodic positive/diversifying selection.

Results of BGM analysis for coevolving sites

Does not show "triangle" linked coevolving sites e.g. site pairs (1,2) and (2,3)

"ProbS1andS2" is the posterior probablity that sites 1 and 2 are not conditionally independent.

Shared subs is the number of substitions shared between a pair of sites.

Figure 5. BGM analysis of your gene of interest found 395 pairs of coevolving sites out of 1167 total sites to be statisically significant (posterior probability threshold 0.5).

Results of aBSREL analysis for episodic diversifying selection on branches

Displaying significant branches and nodes in red.

Figure 6. aBSREL analysis of your gene of interest found 38 of 513 branches to be statisically significant (p-value <= 0.05) for episodic diversifying selection

Which lineages are represented in my alignment?

269 Species in the alignment.
Aim for between 5-10 lineage descriptions.

What am I left with? 6 clade descriptions

Mammalia (perhaps subdivide?)
Osteoglossocephalai = bony fishes
Sauropsida = reptiles
Gymnophiona = amphibians
Erpetoichthys = ropefish (~1% = 2-3 species.)
Batrachia = tailless amphibians (like frogs) (1 specie?)
Semionotiformes = (1 specie?)

Discussion

Is this gene under selection?

Do significant sites between MEME and FEL overlap? They should for positively selected sites, and should not for negatively selected (statisically significant) FEL sites.

Are coevolving sites under positive or negative selection, linked-selection

BUSTEDS

RELAX, how many lineages do I have? Do analysis between branches.

aBSREL
MEME, FEL, SLAC, FUBAR

GARD, subsample to ~30, Discuss recombination

References

HyPhy
Datamonkey
All HyPhy methods
Accessory methods, TN93, MAFFT, IQTREE
Evolution of Viral Genomes: Interplay Between Selection, Recombination, and Other Forces

Supplementary Tables

Table 1. BUSTEDS Fits, test results

Table 2. MEME – Significant sites

Table 3. FEL – Negative Significant sites

Tavke 4. aBSREL

#	Baseline MG94xREV	Baseline MG94xREV omega ratio	Corrected P-value	Full adaptive model	Full adaptive model (non-synonymous subs/site)	Full adaptive model (synonymous subs/site)	LRT	Nucleotide GTR	Rate Distributions	Rate classes	Uncorrected P-value	original name
1	0.271015	0.248128	0.00101124	1.24717	1.16192	0.0852501	23.9811	0.219682	[[0.08351739391025588, 0.814034501673088], [25.78312631442658, 0.185965498326912]]	2	2.05536e-06	nan
2	0.247485	0.402128	0.0224881	379.635	379.548	0.0870347	17.756	0.193016	[[0.1794147285931847, 0.8288530517605159], [9090.000000003736, 0.1711469482394841]]	2	4.68503e-05	nan
3	0.0148508	0.518222	0.000322385	0.175039	0.172598	0.00244112	26.2757	0.0177287	[[0, 0.9744626224056592], [987.8020916848121, 0.02553737759434083]]	2	6.49969e-07	nan
4	0.0688569	1e+10	0.0483007	1.04917	1.04569	0.00347803	16.2193	0.0859847	[[0, 0.8800201943708488], [894.04943259379, 0.1199798056291512]]	2	0.000101472	nan
5	0.147321	0.783096	0.0114947	0.658764	0.639233	0.0195312	19.1077	0.145414	[[0.3838030590330714, 0.8444078195665582], [72.96547749683984, 0.1555921804334418]]	2	2.37494e-05	nan
6	0.0494127	0.248901	0.0391371	2.74932	2.73446	0.0148621	16.6416	0.0494891	[[0.08171724964798617, 0.957908917963494], [1557.699535943612, 0.04209108203650602]]	2	8.20484e-05	nan
7	0.214756	0.410058	0.000978347	0.871563	0.806064	0.0654993	24.051	0.180228	[[0.1095580176944264, 0.8408750960441533], [27.01378932551229, 0.1591249039558467]]	2	1.98448e-06	nan
8	0.216423	0.404559	0.00334964	0.909491	0.84621	0.0632809	21.5789	0.192698	[[0.08907333920270652, 0.8257657450393591], [26.96026079730138, 0.1742342549606409]]	2	6.86401e-06	nan
9	0.0193913	0.467868	0.00343106	2.81498	2.81013	0.00485433	21.5269	0.0181038	[[0.1905853889518029, 0.978009820385468], [9383.731856578368, 0.02199017961453198]]	2	7.04531e-06	nan
10	0.0239495	0.406299	0	0.582777	0.580037	0.00273964	121.617	0.0215566	[[0, 0.9502300085151673], [1517.731423200614, 0.04976999148483274]]	2	0	nan
11	0.0562303	0.445824	0.000189855	0.323557	0.30473	0.0188271	27.3394	0.0539543	[[0.2697852411103061, 0.968801038023459], [176.7157679676714, 0.03119896197654104]]	2	3.81235e-07	nan
12	0.040963	0.863876	0	0.40471	0.40117	0.00353967	126.751	0.040804	[[0.05420569097218374, 0.9502046359232122], [811.0053477596006, 0.04979536407678775]]	2	0	nan
13	0.173598	0.346965	0.0120047	2.73766	2.72766	0.0100021	19.0172	0.134369	[[1, 0.8545620545506069], [663.1141152380948, 0.1454379454493931]]	2	2.48545e-05	nan
14	0.114996	0.809018	0.0028013	1.7344	1.73436	4.28557e-05	21.9431	0.100148	[[1, 0.8556210297601691], [100000, 0.1443789702398309]]	2	5.71693e-06	nan
15	0.122433	0.478445	0.000576403	1.01034	0.995354	0.0149887	25.1094	0.102575	[[0.2539175969811507, 0.8727106483495446], [184.3916331081659, 0.1272893516504554]]	2	1.16681e-06	nan
16	0.00817209	0.340903	0.0347516	0.119071	0.117071	0.00199951	16.8821	0.0080027	[[0, 0.993626957337727], [3277.778847969584, 0.00637304266227301]]	2	7.2702e-05	XM_005749774_1_PREDICTED_Pundamilia_nyererei_cellular_tumor_antigen_p53_like_LOC102203120_mRNA_1
17	0.103901	0.293289	0.00233172	10.3832	10.3375	0.0456859	22.3126	0.0981326	[[0.1357129025907057, 0.9482367361682671], [1557.113665935761, 0.05176326383173291]]	2	4.74892e-06	XM_007952255_1_PREDICTED_Orycteropus_afer_afer_tumor_protein_p53_TP53_mRNA_1
18	0.0457502	0.32037	1.27578e-11	0.241786	0.22455	0.0172363	60.3389	0.0455824	[[0.02364363647801504, 0.9625574726250689], [123.5301901932855, 0.03744252737493114]]	2	2.53131e-14	XM_008010192_2_PREDICTED_Chlorocebus_sabaeus_tumor_protein_p53_TP53_transcript_variant_X1_mRNA_1
19	0.356247	0.320588	7.02686e-07	3.65051	3.53951	0.110996	38.5186	0.312879	[[0.1159697821727439, 0.7769833043336732], [50.61114657575612, 0.2230166956663268]]	2	1.40537e-09	XM_008321788_3_PREDICTED_Cynoglossus_semilaevis_tumor_protein_p53_tp53_mRNA_1
20	0.110145	0.398121	0.00331385	0.673168	0.630454	0.0427146	21.6043	0.107742	[[0.05381489102857581, 0.9132353699648752], [60.12599823633681, 0.0867646300351248]]	2	6.7768e-06	XM_010571169_1_PREDICTED_Haliaeetus_leucocephalus_tumor_protein_p53_TP53_mRNA_1
21	0.0315783	0.366843	2.68195e-08	6.57414	6.56455	0.0095854	45.047	0.0318192	[[0, 0.9731198669707395], [9090.000000003736, 0.02688013302926051]]	2	5.34253e-11	XM_011727613_2_PREDICTED_Macaca_nemestrina_tumor_protein_p53_TP53_mRNA_1
22	0.248904	0.221391	0.0160571	102.609	102.513	0.0962467	18.4343	0.204709	[[0.1563804928982661, 0.9012698341888755], [3847.530319680883, 0.09873016581112448]]	2	3.33135e-05	XM_014019513_1_PREDICTED_Austrofundulus_limnaeus_tumor_protein_p53_tp53_mRNA_1
23	0.121	0.319189	1.87822e-12	4.44712	4.42298	0.024138	64.1631	0.11054	[[0.0168368360727766, 0.885082564787816], [568.7597482656681, 0.114917435212184]]	2	3.71925e-15	XM_014261886_1_PREDICTED_Pseudopodoces_humilis_tumor_protein_p53_TP53_mRNA_1
24	0.176395	0.243335	9.1023e-05	1.9998	1.93635	0.0634534	28.8093	0.15838	[[0.02835874316489893, 0.8674912613583818], [81.9787911678715, 0.1325087386416182]]	2	1.82411e-07	XM_014893382_1_PREDICTED_Sturnus_vulgaris_tumor_protein_p53_TP53_partial_mRNA_1
25	0.135205	0.7309	0	81.0506	81.0302	0.0203742	88.5054	0.134317	[[0.1397099080155256, 0.8439130611686997], [9090.000000003736, 0.1560869388313003]]	2	0	XM_015541817_1_PREDICTED_Panthera_tigris_altaica_tumor_protein_p53_TP53_partial_mRNA_1
26	0.0422313	0.749838	6.26628e-10	7.36183	7.35356	0.00827325	52.5537	0.0416236	[[0.3056022114043056, 0.9651459173578737], [9090.000000003736, 0.03485408264212631]]	2	1.24578e-12	XM_015562573_1_PREDICTED_Myotis_davidii_tumor_protein_p53_TP53_transcript_variant_X1_mRNA_1
27	0.125564	0.678655	0	35.3403	35.3302	0.0101213	192.752	0.124534	[[0.0676211734326852, 0.8629977433488712], [9090.000000003736, 0.1370022566511288]]	2	0	XM_015992323_1_PREDICTED_Peromyscus_maniculatus_bairdii_tumor_protein_p53_Tp53_transcript_variant_X1_mRNA_1
28	0.0714231	0.796249	0	13.9542	13.9481	0.00612411	157.307	0.0720728	[[0, 0.9106059867613868], [9090.000000003736, 0.08939401323861318]]	2	0	XM_016931470_2_PREDICTED_Pan_troglodytes_tumor_protein_p53_TP53_transcript_variant_X1_mRNA_1
29	0.0594683	0.427825	5.62883e-14	0.561761	0.551066	0.0106943	70.5412	0.0498929	[[0.1259844837537699, 0.9514527920641109], [376.2254588321816, 0.04854720793588907]]	2	1.11022e-16	XM_025195179_1_PREDICTED_Alligator_sinensis_tumor_protein_p53_TP53_transcript_variant_X1_mRNA_1
30	0.041931	0.258989	0.0219854	0.101613	0.0799033	0.0217093	17.8051	0.0408591	[[0.03863496214427223, 0.9690592305995114], [41.23128517951871, 0.03094076940048862]]	2	4.57078e-05	XM_028780049_1_PREDICTED_Grammomys_surdaster_tumor_protein_p53_Tp53_transcript_variant_X1_mRNA_1
31	0.0610096	0.310105	0.00420115	5.10637	5.07529	0.0310796	21.1196	0.0621175	[[0.1018540946120266, 0.9626603774309264], [1557.699906332703, 0.03733962256907364]]	2	8.64434e-06	XM_030474375_1_PREDICTED_Strigops_habroptila_tumor_protein_p53_TP53_transcript_variant_X1_mRNA_1
32	0.0550798	0.651481	0.00619516	0.335227	0.323262	0.0119644	20.3421	0.0548318	[[0.2411625338720781, 0.9514469608672388], [193.8137073349213, 0.04855303913276121]]	2	1.27735e-05	XM_030548110_1_PREDICTED_Gopherus_evgoodei_tumor_protein_p53_TP53_mRNA_1
33	0.0355974	1.00236	0	25.751	25.7489	0.00206024	87.036	0.0354803	[[0.7066674874553129, 0.9554163256482412], [100000, 0.04458367435175881]]	2	0	XM_030861565_1_PREDICTED_Globicephala_melas_tumor_protein_p53_TP53_transcript_variant_X1_mRNA_1
34	0.120016	0.254143	0.022593	0.893713	0.83993	0.0537829	17.7426	0.108033	[[0.09255911594996524, 0.9144690076952635], [64.15460824206325, 0.08553099230473649]]	2	4.7167e-05	XM_030970056_1_PREDICTED_Camarhynchus_parvulus_tumor_protein_p53_TP53_transcript_variant_X1_mRNA_1
35	0.0242619	0.599177	0.000241366	0.0477902	0.0401435	0.00764665	26.8568	0.0250599	[[0.2009160516548929, 0.9798753216487776], [83.28854940137336, 0.02012467835122245]]	2	4.85646e-07	XM_032280430_1_PREDICTED_Sapajus_apella_tumor_protein_p53_TP53_transcript_variant_X1_mRNA_1
36	0.0515846	0.500838	0.000548347	15.0164	15.0013	0.0150812	25.2129	0.0499321	[[0.1917560263656985, 0.9609786199719969], [9090.000000003736, 0.03902138002800315]]	2	1.10777e-06	XM_032794536_1_PREDICTED_Chelonoidis_abingdonii_tumor_protein_p53_TP53_mRNA_1
37	0.213434	0.37986	3.69073e-08	95.6181	95.564	0.0540627	44.4054	0.193231	[[0.1554275170793705, 0.8360599805867974], [3846.116864262084, 0.1639400194132026]]	2	7.36673e-11	XM_035135727_1_PREDICTED_Zootoca_vivipara_tumor_protein_p53_TP53_transcript_variant_X2_mRNA_1
38	0.0430373	0.588996	0	0.65763	0.649333	0.00829716	87.2536	0.0433632	[[0.2025151800612972, 0.9628558985353769], [746.4563181259871, 0.0371441014646231]]	2	0	XM_041664596_1_PREDICTED_Microtus_oregoni_tumor_protein_p53_Tp53_transcript_variant_X1_mRNA_1

Phylogenetic tree analysis, histogram of branch lengths and what is the total tree length?

AlignmentProfiler (Skip for now)

v1 https://colab.research.google.com/drive/1JWvp9zTEqCslH5P2_9UhwQllmOJOGAF9?usp=sharing

v2 https://colab.research.google.com/drive/1fNjBYkIOH9DFD8hdmXJTekj8Hzg-g3Jl?usp=sharing

Left over (can delete)

BUSTEDS Unconstrained model	BUSTEDS Constrained model

Figure 1. BUSTEDS found evidence found evidence (LRT, p-value ≤ 0.05) of gene-wide episodic diversifying selection in the selected test branches of your phylogeny.