Analysis of Positive Selection in Aspergillus

In this repository we describe the workflow followed to process and analyze the data of the study "Aspergillus fumigatus versus Genus Aspergillus: Conservation, adaptive evolution and specific virulence genes".

Issues & Contacts

Please note that these scripts were not designed to function as a fully-automated pipeline, but rather as a series of individual steps with extensive manual quality control between them. It will therefore not be straightforward to run all steps smoothly in one go. Feel free to contact me (shishir.gupta@uni-wuerzburg.de) or Prof. Dr. Thomas Dandekar (dandekar@biozentrum.uni-wuerzburg.de) if you run into any issue.

Requirements

Perl, BioPerl, Blast, T-Coffeee, Pal2Nal, Gblocks, Hyphy, RaxML, PAML. All the programs should be installed and path should be added in the bash profile (Unix OS).

1. Sequences

All the amino acid sequences used can be downloaded from here and nucleotide sequences can be downloaded from here.

2. Orthology analysis

Orthology were computed using standerd protocol of OrthoMCL. Please find the access link of software here and protocol for computations here.

The resulted cluster from orthology analysis can be obtained here. Single copy ortholog clusters were extracted by the bash script extract_single_copy_orthologs.sh.

3. Alignments

$ perl run_tcoffee.pl -i protein_cluster_dir

$ perl run_pal2nal.pl -p protein_alignment_dir -d gene_cluster_dir -c directory_codon_alignments

Removal of Unreliable Alignment Regions

$ perl run_gblocks.pl -i directory_codon_alignments

Nucleotide alignment of single copy orthologs can be obtained from here.

4. Recombination

Create a text file named as input_path.txt which should contain the full path to each tested alignment file per line. Place the file in the same directory as the alignment files and execute the following command on the terminal.

$ mpirun -np 4 HYPHYMPI run_GARD.bf

$ perl run_GARDProcessor.pl -i gard_directory -t GARDProcessor_temp.bf -p full_path_to_working_ directory

$ perl summary_breakpoint.pl -i gard_dir -o breakpoint.tab

5. Evolutionary analyses

Generation of species tree

Keep script concat_alignments.pl in the directory with amino acid sequence alignment of single copy orthologs

$ perl concat_alignments.pl

$ perl degapper.pl -in concat_alignments.fa -limit 0.5

$ raxmlHPC-PTHREADS-AVX -f a -T 2 -p 12345 -m PROTGAMMAAUTO -x 12345 -# 10000 -s concat_alignments.fa.proc -m PROTGAMMAAUTO -n T20

For null model

$ perl run_codeml_BS_Null.pl -s phylip -t species_tree.txt -o M1a > run.log &

$ perl parse_codeml_BS_H0.pl M1a codeml-bs-M1a.txt

For alternative model

$ perl run_codeml_BS_modelA.pl -s phylip -t species_tree.txt -o M2a > run.log &

$ perl parse_codeml_BS_H1.pl M2a codeml-bs-M2a.txt

Statistics

$ perl combineM1-M2.pl codeml-bs-M2a.txt codeml-bs-M1a.txt > gene.list

$ perl statistics-chi2-df1-pvalue.pl gene.list > combineM1aM2a_addPvalue_gene.list.txt

$ perl calculating-qvalue-BY.pl combineM1aM2a_addPvalue_gene.list.txt > combinedM1aM2a_addQvalue_gene.list.txt

Data files

Accession number: good quality CDS

Please click here to access the 2,08,055 CDS sequence file in Zip format.

Accession number: good quality proteins

Please click here to access the 2,08,055 amino acid sequence file in Zip format.

Additional files

Additional data and material can be found here in a single zip file. Sequence alighment of Single Copy Orthologs can be obtained from here

Additional file 1: Supplementary figures -

Figure S1: Proteins-protein interactions (PPIs) map. PPIs between A. fumigatus proteins coded by positively selected genes (PSGs) and human proteins. Orange - A. fumigatus proteins and blue - human proteins.

Figure S2: Gene Ontology (GO) overrepresentation analysis. Biological process (GO) significantly overrepresented in human proteins targeted by A. fumigatus virulence related proteins coded by multi-copy category positively selected genes (PSGs).

Figure S3: A. fumigatus and A. fischeri genome synteny map. Image was generated using the Mauve genome alignment tool. Upper and lower lines of the genomes correspond to A. fumigatus and A. fischeri respectively. Red vertical bars indicate concatenated chromosomal boundaries. Color-coded syntenic blocks indicate conserved segments (LCBs; Locally Collinear Blocks) identified by Mauve (minimum LCB weight = 999). Plots of sequence similarity are shown within each syntenic block. Regions with no color indicate no detectable homology between the two genomes with the settings used in Mauve. Unmatched regions (white area) within an LCBs indicate the presence of strain-specific sequence. The connecting lines between blocks indicate the location of each block in two genomes. Homologous regions with possible rearrangements are shown in first row.

Additional file 1: data file 1:

Clusters of orthologues identified by OrthoMCL (inflation index: 1.5). Abbreviations - abra : Aspergillus brasiliensis, acar : A. carbonarius, acla : A. clavatus, afis : A. fischeri, afla : A. flavus, afum : A. fumigatus, agla : A. glaucus, akaw : A. kawachii, alue : A. luchuensis, anid : A. nidulans, anig : A. niger, aora : A. oryzae, asyd : A. sydowii, ater : A. terreus, atub : A. tubingenesis, aver : A. versicolor, awen : A. wentii, azon : A. zonatus.

Additional file 1: data file 2:

Core genome of Aspergillus. The file consists of all the clusters containing at least one protein from all the analysed 18 species. Abbreviations - abra : Aspergillus brasiliensis, acar : A. carbonarius, acla : A. clavatus, afis : A. fischeri, afla : A. flavus, afum : A. fumigatus, agla : A. glaucus, akaw : A. kawachii, alue : A. luchuensis, anid : A. nidulans, anig : A. niger, aora : A. oryzae, asyd : A. sydowii, ater : A. terreus, atub : A. tubingenesis, aver : A. versicolor, awen : A. wentii, azon : A. zonatus.

Additional file 1: data file 3:

A. fumigatus and A. fischeri orthology analysis (Inparanoid output).

Additional file 2 –

Table S1. Conservancy of A. fumigatus specific genes in other A. fumigatus strains

Table S2. Basic genomic information of all eighteen aspergilli

Table S3. GO over-representation analysis of single copy orthologs in the three GO categories (i.e. ‘Biological Processes’, BP; ‘Molecular Function’, MF; ‘Cellular Component’, CC)

Table S4. Quantitative summary of orthology assignments

Table S5. GO over-representation analysis of recombination breakpoint containing clusters in the three GO categories (i.e. ‘Biological Processes’, BP; ‘Molecular Function’, MF; ‘Cellular Component’, CC)

Table S6. Pathway overrepresentation of host proteins interacting with A. fumigatus positively selected proteins

Table S7. Gene Ontology (GO) overrepresentation of host proteins interacting with A. fumigatus positively selected proteins

Table S8. Positively selected genes and KOG annotations

Table S9. Positively selected multi-gene family virulence genes identified in A. fumigatus

Table S10. Genes from top three GO-biological processes with reference to ortholog relation to amoeba species A. castellanii and D. discoideum

Table S11. Positively selected genes and their expression in hypoxia conditions

Table S12. A. fumigatus positively selected hub proteins

Table S13. Degree of A. fumigatus positively selected proteins involved in host interactions

Table S14. Positively selected genes identified in A. fischeri

Table S15. Recombination breakpoints in alignments

Additional file 3: Supplemental methods