DupGen_finder

The DupGen_finder was developed to identify different modes of duplicated gene pairs. MCScanX algorithm was incorporated in this pipeline.

The schematic diagram of DupGen_finder pipeline

Figure 1: The flowchart of DupGen_finder pipeline ## Contents * [Dependencies](#dependencies) * [Installation](#installation) * [Preparing input files](#preparing-input-files) * [Running](#running) * [Result Files](#result-files) * [Citation](#citation) ## Dependencies - [Perl](https://www.perl.org) ## Installation ```bash cd ~/software # or any directory of your choice git clone https://github.com/qiao-xin/DupGen_finder.git cd DupGen_finder make chmod 775 DupGen_finder.pl chmod 775 DupGen_finder-unique.pl chmod 775 set_PATH.sh source set_PATH.sh ``` Test you can run DupGen_finder: ```bash DupGen_finder.pl ``` DupGen_finder should print its 'help' text. **\*\*Note\*\*** DupGen_finder software package includes a custom MCScanX algorithm which can output sorted `gff` files such as `Ath.gff.sorted`, and is slightly different from the original MCScanX algorithm implemented in [MCScanX](http://chibba.pgml.uga.edu/mcscan2/) software package. ## Preparing input files Pre-computed BLAST results (-outfmt 6) and gene location information (GFF format) are required for running DupGen_finder successfully. 1. For the target genome in which gene duplicaiton modes will be classified, please prepare two input files: - ```target_species.gff```, a gene position file for the target species, following a tab-delimited format. For example, "Ath.gff". - ```target_species.blast```, a blastp output file (-outfmt 6) for the target species (self-genome comparison). For example, "Ath.blast". 2. For the outgroup genome, please prepare two input files: - ```[target_species]_[outgroup_species].gff```, a gene position file for the target_species and outgroup_species, following a tab-delimited format. - ```[target_species]_[outgroup_species].blast```, a blastp output file (-outfmt 6) between the target and outgroup species (cross-genome comparison). For example, assuming that you are going to classify gene duplication modes in *Arabidopsis thaliana* (Ath), using *Nelumbo nucifera* (Nnu) as outgroup, you need to prepare 4 input files: ```Ath.gff```,```Ath.blast```, ```Ath_Nnu.gff```, ```Ath_Nnu.blast``` **gff file** ```Ath.gff``` is in the following format (tab separated): ``` Species_abbrev-Chr_ID gene_ID start_position end_position ``` The data in ```Ath.gff``` looks like this (tab separated): ``` Ath-Chr1 AT1G01010.1 3631 5899 Ath-Chr1 AT1G01020.1 5928 8737 Ath-Chr1 AT1G01030.1 11649 13714 Ath-Chr1 AT1G01040.2 23416 31120 Ath-Chr1 AT1G01050.1 31170 33153 ``` The below command can be used to creat ```Ath_Nnu.gff```: ```bash cat Ath.gff Nnu.gff >Ath_Nnu.gff ``` The data in ```Ath_Nnu.gff``` looks like this (tab separated): ``` Ath-Chr1 AT1G01010.1 3631 5899 Ath-Chr1 AT1G01020.1 5928 8737 ... Nnu-megascaffold_32 NNU_00001-RA 57481 63788 Nnu-megascaffold_32 NNU_00002-RA 32491 41125 ... ``` **blast file** ```Ath.blast``` is in the following format: ``` query acc.ver, subject acc.ver, % identity, alignment length, mismatches, gap opens, q. start, q. end, s. start, s. end, evalue, bit score ``` The data in ```Ath.blast``` looks like this (tab separated): ``` ATCG00500.1 ATCG00500.1 100.00 488 0 0 1 488 1 488 0.0 932 ATCG00510.1 ATCG00510.1 100.00 37 0 0 1 37 1 37 2e-19 73.9 ATCG00280.1 ATCG00280.1 100.00 473 0 0 1 473 1 473 0.0 876 ATCG00890.1 ATCG01250.1 100.00 389 0 0 1 389 1 389 0.0 660 ATCG00890.1 ATCG00890.1 100.00 389 0 0 1 389 1 389 0.0 660 ``` Here is the typical parameter setting for generating the xyz.blast file: The example file ```Ath.pep``` contains the whole genome protein sequences (FASTA format) of Arabidopsis. - For BLAST software ```bash # Create a reference database makeblastdb -in Ath.pep -dbtype prot -title Ath -parse_seqids -out Ath # Align protein query sequences against the reference database blastp -query query_file -db database -evalue 1e-10 -max_target_seqs 5 -outfmt 6 -out xyz.blast # For example blastp -query Ath.pep -db Ath -evalue 1e-10 -max_target_seqs 5 -outfmt 6 -out Ath.blast ``` - For DIAMOND software ```bash # Create a reference database diamond makedb --in Ath.pep -d Ath # Align protein query sequences against the reference database diamond blastp -d Ath -q Ath.pep -o Ath.blast -p 20 --sensitive --max-target-seqs 5 --evalue 1e-10 --quiet ``` --- **NOTE: All above input files should be stored under the same folder (the "-i" option). For more parameters please see below.** ## Running Run the following command to get help information about **DupGen_finder**: ```bash DupGen_finder.pl ``` This command will print a full list of options: ``` Usage: DupGen_finder.pl -i data_directory -t target_species -c outgroup_species -o output_directory ##################### Optional: -a 1 or 0(are segmental duplicates ancestral loci or not? default: 1, yes) -d number_of_genes(maximum distance to call proximal, default: 10) ##################### The following are optional MCScanX parameters: -k match_score(cutoff score of collinear blocks for MCScanX, default: 50) -g gap_penalty(gap penalty for MCScanX, default: -1) -s match_size(number of genes required to call a collinear block for MCScanX, default: 5) -e e_value(alignment significance for MCScanX, default: 1e-05) -m max_gaps(maximum gaps allowed for MCScanX, default: 25) -w overlap_window(maximum distance in terms of gene number, to collapse BLAST matches for MCScanX, default: 5) ``` A typical command to identify different modes of duplicated gene pairs in a given species could look like this: ```bash DupGen_finder.pl -i data -t Ath -c Nnu -o results ``` Here, **DupGen_finder** attempts to identify the different modes of duplicated gene pairs in *A.thaliana* by using *N.nucifera* as outgroup. All required data files should be stored under this directory ```data```. The output files will be stored under this directory ```results```. For more details please see below. Ath: *A.thaliana*, Nnu: *N.nucifera*. **Note**: We recommend that the "data_directory" or "output_directory" should be given a full path. For example, ```/home/the_path_to_your_data_directory/``` ### *DupGen_finder-unique* Moreover, to eliminate redundant duplicate genes among different modes, we provide a stricter version of **DupGen_finder** named **DupGen_finder-unique** by which each duplicate gene was assigned to a unique mode after all of the duplicated gene pairs were classified into different gene duplication types. The priority of the duplicate genes is as follows: WGD > tandem > proximal > transposed > dispersed. ```bash DupGen_finder-unique.pl -i data -t Ath -c Nnu -o results ``` ## Result Files ### 1 - Duplicated gene pairs: - Ath.wgd.pairs - Ath.tandem.pairs - Ath.proximal.pairs - Ath.transposed.pairs - Ath.dispersed.pairs These files includes duplicated gene pairs derived from five modes of gene duplication, including WGD (**Ath.wgd.pairs**), tandem duplication (**Ath.tandem.pairs**), proximal duplication (**Ath.proximal.pairs**), transposed duplication (**Ath.transposed.pairs**), dispersed duplication (**Ath.dispersed.pairs**). The gene pairs contained in these files looks like this (tab separated): ``` Duplicate 1 Location Duplicate 2 Location E-value AT1G01010.1 Ath-Chr1:3631 AT4G01550.1 Ath-Chr4:673862 5e-52 AT1G01020.1 Ath-Chr1:5928 AT4G01510.1 Ath-Chr4:642733 5e-74 AT1G01030.1 Ath-Chr1:11649 AT1G13260.1 Ath-Chr1:4542168 3e-45 AT1G01050.1 Ath-Chr1:31170 AT3G53620.1 Ath-Chr3:19880504 6e-126 AT1G01060.1 Ath-Chr1:33666 AT5G17300.1 Ath-Chr5:5690227 3e-30 ``` ### 2 - Ath.singletons It includes genes that have no homologous genes within target species. ``` GeneID Location AT2G32600.1 Ath-Chr2:13833545 AT5G11810.1 Ath-Chr5:3808720 AT4G16610.1 Ath-Chr4:9354321 AT1G66520.1 Ath-Chr1:24816128 AT1G51110.1 Ath-Chr1:18935329 ``` ### 3 - Ath.stats The number of duplicated gene pairs derived from different modes. ``` Types NO. of gene pairs WGD-pairs 4352 TD-pairs 2063 PD-pairs 788 TRD-pairs 4447 DSD-pairs 16130 ``` ### 4 - Collinearity files - Ath.collinearity - Ath_Nnu.collinearity ## Citation *[Qiao X, Li Q, Yin H, Qi K, Li L, Wang R, Zhang S, Paterson AH: Gene duplication and evolution in recurring polyploidization–diploidization cycles in plants. Genome Biology 2019, 20:38.](https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1650-2)*