Here are the scripts and documents to conduct strand and WGD aware syntenic gene identification for a pair of genomes using the longest path algorithm implemented in AnchorWave. We currently provide three visualization methods for syntenic results.
You can simple by the following command get this software in a independent conda envirment. This is a beta version, so we haven't uploaded it to bioconda yet.
conda install xiaodli::quota_anchor
quota_Anchor -h
usage: quota_Anchor [-h] [-v] {pre_col,col,get_chr_length,dotplot,circle,line_2,line_proali} ...
Conduct strand and WGD aware syntenic gene identification for a pair of genomes using the longest path algorithm implemented in AnchorWave:
options:
-h, --help show this help message and exit
-v, --version show program's version number and exit
gene collinearity analysis:
{pre_col,col,get_chr_length,dotplot,circle,line_2,line_proali}
pre_col Get longest protein file from gffread result and input file for gene collinearity analysis
col Get gene collinearity result file
get_chr_length Get chromosome length and name info from fai file
dotplot Collinearity result visualization
circle Collinearity result visualization
line_2 Collinearity result visualization
line_proali Anchors file from AnchorWave proali visualization
Here is an example to identify syntenic genes between maize and sorghum. The maize lineage has undergone a whole genome duplication (WGD) since its divergence with sorghum, but subsequent chromosomal fusions resulted in these species having the same chromosome number (n = 10). AnchorWave can allow up to two collinear paths for each sorghum anchor while one collinear path for each maize anchor.
Working directory structure are as follows. We may give you an option to create this directory later, but you will need to do it yourself now.
├── length_file
│ └── get_length.conf
├── raw_data
│ ├── Sorghum_bicolor.Sorghum_bicolor_NCBIv3.57.gff3
│ ├── Sorghum_bicolor.Sorghum_bicolor_NCBIv3.dna.toplevel.fa
│ ├── Zm-B73-REFERENCE-NAM-5.0.fa
│ └── Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3
└── sb_zm
└── config_file
├── circle.conf
├── collinearity.conf
├── dotplot.conf
├── line.conf
└── pre_collinearity.conf
Put the genome and gff file into the sb_zm/raw_data directory.
wget https://download.maizegdb.org/Zm-B73-REFERENCE-NAM-5.0/Zm-B73-REFERENCE-NAM-5.0.fa.gz
wget https://download.maizegdb.org/Zm-B73-REFERENCE-NAM-5.0/Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3.gz
wget https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-57/fasta/sorghum_bicolor/dna/Sorghum_bicolor.Sorghum_bicolor_NCBIv3.dna.toplevel.fa.gz
wget https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-57/gff3/sorghum_bicolor/Sorghum_bicolor.Sorghum_bicolor_NCBIv3.57.gff3.gz
gunzip *gz
This includes four steps(implemented in "quota_Anchor pre_col")
- Extract and translate protein sequences from genome sequences and annotations
- Identify and extract the longest protein sequence encoded by each gene
- Protein sequence alignment using DIAMOND or conduct protein sequence alignment using BLASTp
- Put the gene strand information and the blast result into a single file
| Header | Parameter | Description |
|---|---|---|
| [gffread] | ref_genome_seq | Please provide e path to your reference FASTA file |
| ref_gff_file | Please provide a path to your reference GFF file | |
| output_ref_pep_seq | Please type a filename for the reference protein sequence file that the software will generate | |
| query_genome_seq | Please provide a path to your Query FASTA file | |
| query_gff_file | Please provide a path to your Query GFF file | |
| output_query_pep_seq | Please type a filename for a query protein sequence file that the software will generate | |
| use_S_parameter | When using gffread -y option, whether the -S parameter is used, which means using '*' instead of '.' as the stop codon for translation(Not recommended to modify, default:True) | |
| [longest_pep] | out_ref_longest_pep_name | Please type a filename for longest protein sequence file that software will generate |
| out_query_longest_pep_name | Please type a filename for longest protein sequence file that software will generate | |
| [align] | align | Protein sequence alignment using DIAMOND or BLASTp(please type: diamond or blastp) |
| [diamond] | database_name | Please type a diamond-blastp database Path/Name that software will generate |
| output_blast_result | Please type a diamond-blastp result Path that software will generate | |
| max_target_seqs | Maximum number of target sequences to report alignments for diamond-blastp | |
| evalue | Maximum e-value to report alignments | |
| [blastp] | database_name | Please type a blastp database Path/Name that software will generate |
| dtype | Database date type(please type:prot) | |
| output_blast_result | Please type a blastp result Path that software will generate | |
| evalue | Maximum e-value to report alignments | |
| max_target_seqs | Maximum number of target sequences to report alignments for diamond-blastp | |
| thread | Number of CPU threads | |
| outfmt | Please type 6(BLAST tabular) | |
| [combineBlastAndStrand] | out_file | Please type a filename for a longest protein sequence file that software will generate |
| bitscore | Filter by blast minimum bitscore(default:100) | |
| align_length | Filter by blast minimum alignment length(default:0) | |
Put the following information into the sb_zm/config_file/pre_collinearity.conf file.
[gffread]
ref_genome_seq = ../raw_data/Sorghum_bicolor.Sorghum_bicolor_NCBIv3.dna.toplevel.fa
ref_gff_file = ../raw_data/Sorghum_bicolor.Sorghum_bicolor_NCBIv3.57.gff3
output_ref_pep_seq = sb.p.fa
query_genome_seq = ../raw_data/Zm-B73-REFERENCE-NAM-5.0.fa
query_gff_file = ../raw_data/Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3
output_query_pep_seq = zm.p.fa
# The next line is the description of the S parameter of gffread(https://github.com/gpertea/gffread), you need to set True in general.
# -S for -y option, use '*' instead of '.' as stop codon translation
use_S_parameter = True
[longest_pep]
out_ref_longest_pep_name = sorghum.protein.fa
out_query_longest_pep_name = maize.protein.fa
[align]
align= diamond
[diamond]
# use ref protein seq construct database
database_name = sorghum.db
output_blast_result = sorghum.maize.diamond.blastp
max_target_seqs = 20
evalue = 1e-10
[blastp]
database_name = sorghum.blastp.db
dtype = prot
output_blast_result = sorghum.maize.blastp
evalue = 1e-10
max_target_seqs = 20
thread = 6
outfmt = 6
[combineBlastAndStrand]
out_file = sb_zm.table
bitscore = 100
align_length = 0
You can run this command in the sb_zm directory.
quota_Anchor pre_col -c ./config_file/pre_collinearity.conf
| [AnchorWave] | R | The R value indicates the maximum number of occurrences of a reference gene in the collinearity file |
| Q | The Q value indicates the maximum number of occurrences of a query gene in the collinearity file | |
| maximum_gap_size | maximum gap size for chain | |
| collapse_blast_matches | Specify whether to collapse blast mathches in the input file (default: 0) Options: 0 don't collapse blast matches; 1 or any other integer to collapse them. | |
| overlap_window | When -m is set to collapse blast matches(1 or any other integer), specify the maximum distance(overlap window) allowed between two homologous gene pairs before they are considered for deletion(default: 5) This parameter is ignored if -m is not set to collapse overlap window(set 0). | |
| input_file_name | quotaAnchor pre_col command module output file path(Please type combineBlastAndStrand.out_file 's value) | |
| output_coll_name | Please type a syntenic result filename that software will generate | |
Put the following information into the sb_zm/config_file/collinearity.conf file and running colllinearity analysis.
[AnchorWave]
# The R value indicates the maximum number of occurrences of a gene in the collinearity file, and Q means the same as R.
# For maize and sorghum, maize has undergone an additional whole-genome duplication compared to sorghum.
# If sorghum is used as a reference, you can set R to 2 and Q to 1.
R = 2
Q = 1
maximum_gap_size = 25
collapse_blast_matches = 0
overlap_window = 5
input_file_name = sb_zm.table
output_coll_name = sb_zm.table.collinearity
quota_Anchor col -c ./config_file/collinearity.conf
The sb_zm.table could be visualized via the following R code:
library(ggplot2)
changetoM <- function ( position ){
position=position/1000000;
paste(position, "M", sep="")
}
data =read.table("sb.zm.table")
data$strand = data$V6==data$V12
data[which(data$strand),]$strand = "+"
data[which(data$strand==FALSE),]$strand = "-"
data = data[which(data$V8 %in% c("1", "2", "3", "4", "5", "6", "7", "8", "9", "10")),]
data = data[which(data$V2 %in% c("chr1", "chr2", "chr3", "chr4", "chr5", "chr6", "chr7", "chr8", "chr9", "chr10")),]
data$V8 = factor(data$V8, levels=c("1", "2", "3", "4", "5", "6", "7", "8", "9", "10"))
data$V2 = factor(data$V2, levels=c("chr1", "chr2", "chr3", "chr4", "chr5", "chr6", "chr7", "chr8", "chr9", "chr10"))
plot = ggplot(data=data, aes(x=V10, y=V4))+geom_point(size=0.5, aes(color=strand))+facet_grid(V2~V8, scales="free", space="free" )+ theme_grey(base_size = 30) +
labs(x="sorghum", y="maize")+scale_x_continuous(labels=changetoM) + scale_y_continuous(labels=changetoM) +
theme(axis.line = element_blank(),
panel.background = element_blank(),
panel.border = element_rect(fill=NA,color="black", linewidth=0.5, linetype="solid"),
axis.text.y = element_text( colour = "black"),
legend.position='none',
axis.text.x = element_text(angle=300, hjust=0, vjust=1, colour = "black") )
png("sorghum.maize.table.png" , width=2000, height=1500)
plot
dev.off()
This file of sb_zm.table.colinearity could be visualized via the following R code:
library(ggplot2)
changetoM <- function ( position ){
position=position/1000000;
paste(position, "M", sep="")
}
data = read.table("sb_zm.table.collinearity", header=T)
data = data[which(data$refChr %in% c("1", "2", "3", "4", "5", "6", "7", "8", "9", "10")),]
data = data[which(data$queryChr %in% c("chr1", "chr2", "chr3", "chr4", "chr5", "chr6", "chr7", "chr8", "chr9", "chr10")),]
data$refChr = factor(data$refChr, levels=c("1", "2", "3", "4", "5", "6", "7", "8", "9", "10"))
data$queryChr = factor(data$queryChr, levels=c("chr1", "chr2", "chr3", "chr4", "chr5", "chr6", "chr7", "chr8", "chr9", "chr10"))
plot = ggplot(data=data, aes(x=queryStart, y=referenceStart))+geom_point(size=0.5, aes(color=strand))+facet_grid(refChr~queryChr, scales="free", space="free" )+
theme_grey(base_size = 30) +
labs(x="maize", y="sorghum")+scale_x_continuous(labels=changetoM) + scale_y_continuous(labels=changetoM) +
theme(axis.line = element_blank(),
panel.background = element_blank(),
panel.border = element_rect(fill=NA,color="black", linewidth=0.5, linetype="solid"),
axis.text.y = element_text( colour = "black"),
legend.position='none',
axis.text.x = element_text(angle=300, hjust=0, vjust=1, colour = "black") )
png("sorghum.maize.colinearity.png" , width=2000, height=1500)
plot
dev.off()
| [length] | fai_file | species1.fai, species2.fai, species3.fai, species4.fai |
| gff_file | species1.gff3, species2.gff3, species3.gff3, species4.gff3 | |
| select_fai_chr_startswith | number,CHR,chr,Chr:number,CHR,chr,Chr:number,CHR,chr,Chr:number,CHR,chr,Chr | |
| length_file | This module will output species1.txt, species2.txt, species3.txt, species4.txt |
Put the following information into the sb_zm/length_file/get_length.conf file
# In the process of quotaAnchor pre_col, you can get fai file.
# By fai file and raw GFF file , you can get length information.
# The maize length information example file are as follows.
#chr length total_gene
#chr1 308452471 5892
#chr2 243675191 4751
#chr3 238017767 4103
#chr4 250330460 4093
#chr5 226353449 4485
#chr6 181357234 3412
#chr7 185808916 3070
#chr8 182411202 3536
#chr9 163004744 2988
#chr10 152435371 2705
# select_fai_chr_startswith parameter:
# number: software selects chromosome name starting with number then count the chromosome length.
# chr: software selects chromosome name starting with chr string then count the chromosome length.
# Chr: software selects chromosome name starting with Chr string then count the chromosome length.
[length]
fai_file = ../raw_data/Sorghum_bicolor.Sorghum_bicolor_NCBIv3.dna.toplevel.fa.fai, ../raw_data/Zm-B73-REFERENCE-NAM-5.0.fa.fai
gff_file = ../raw_data/Sorghum_bicolor.Sorghum_bicolor_NCBIv3.57.gff3, ../raw_data/Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3
# By default, the first column of the lines starting with chr or Chr or CHR in the fai file are extracted for plotting.
select_fai_chr_startswith = number,CHR,chr,Chr:number,CHR,chr,Chr
length_file = sb_length.txt, zm_length.txt
quota_Anchor get_chr_length -c get_length.conf
| [circle] | collinearity | Gene collinearity file |
| ref_length | Reference Species length file | |
| query_length | Query Species length file | |
| ref_prefix | Reference Species chromosome prefix(two letters are better) | |
| query_prefix | Query Species chromosome prefix(two letters are better) | |
| font_size | Text font size(you can enter /you/conda/path/envs/envs_name/lib/python3.12/site-packages/quota_anchor/ modify circle.py 's other parameter) | |
| savefig | Specify the file name to save |
Put the following information into the sb_zm/length_file/circle.conf file
# support two species
[circle]
collinearity = sb_zm.table.collinearity
ref_length = ../length_file/sb_length.txt
query_length = ../length_file/zm_length.txt
ref_prefix = sb-
query_prefix = zm-
font_size = 7
savefig = sb_zm.circle.png
quota_Anchor circle -c ./config_file/circle.conf
| [line] | collinearity | Gene collinearity file, e.g. species1_species2.collinearity, species2_species3.collinearity, species3_species4.collinearity |
| length_file | Species length file, e.g. species1_length.txt, species2_length.txt, species3_length.txt, species4_length.txt | |
| prefix | Species prefix, e.g. species1, species2, species3, species4 | |
| remove_chromosome_prefix | Remove chromosome prefix in the result plot (default: chr,CHR,Chr) | |
| text_font_size | Adjust the text font size in the picture | |
| savefig | Specify the file name to save |
Put the following information into the sb_zm/length_file/line.conf file
#Figure from bottom to top (ref:species1, query:species2, ref:species2,query:species3, ref:species3,query:species4 ....)
[line]
collinearity = sb_zm.table.collinearity
length_file = ../length_file/sb_length.txt, ../length_file/zm_length.txt
prefix = Sorghum, Maize
remove_chromosome_prefix = chr,CHR,Chr
text_font_size = 8
savefig = sb_zm.line.png
quota_Anchor line -c ./config_file/line.conf
Put the following information into the sb_zm/length_file/dotplot.conf file
# set(width=1500, heigth=1200) works well[for maize(query) vs sorghum_bicolor(ref)]
[dotplot]
input_file = sb_zm.table
ref_length = ../length_file/sb_length.txt
query_length = ../length_file/zm_length.txt
type = order
query_name = Maize
ref_name = Sorghum bicolor
plotnine_figure_width=1500
plotnine_figure_height=1200
filename= sb_zm.order.table.png
quota_Anchor dotplot -c ./config_file/dotplot.conf
Put the following information into the sb_zm/length_file/dotplot.conf file
# set(width=1500, heigth=1200) works well[for maize(query) vs sorghum_bicolor(ref)]
[dotplot]
input_file = sb_zm.table.collinearity
ref_length = ../length_file/sb_length.txt
query_length = ../length_file/zm_length.txt
type = order
query_name = Maize
ref_name = Sorghum bicolor
plotnine_figure_width=1500
plotnine_figure_height=1200
filename= sb_zm.table.collinearity.png
quota_Anchor dotplot -c ./config_file/dotplot.conf
Put the following information into the sb_zm/length_file/line.conf file
# Figure from bottom to top (ref:species1, query:species2, ref:species2,query:species3, ref:species3,query:species4)
[line]
collinearity = oryza.sorghum.table.collinearity, sorghum.maize.table.collinearity, zm_sv/maize.setaria.table.collinearity
length_file = os_length.txt, sb_length.txt, zm_length.txt, sv_length.txt
prefix = Oryza setaria, Sorghum bicolor, Maize, Setaria viridis
remove_chromosome_prefix = chr,CHR,Chr
text_font_size = 7
savefig = os_sb_zm_sv.line.png
quota_Anchor line_2 -c line.conf
Only identify and extract the longest protein sequence encoded by each gene.
Put the following information into the pre_collinearity.conf file
[gffread]
genome_seq = species1.fa, species2.fa, species3.fa, species4.fa
gff_file = species1.gff3, species2.gff3, species3.gff3, species4.gff3
out_pep_seq = species1.p.fa, species2.p.fa, species3.p.fa, species4.p.fa
# The next line is the description of the S parameter of gffread(https://github.com/gpertea/gffread), you need to set True in general.
# -S for -y option, use '*' instead of '.' as stop codon translation
use_S_parameter = True
[longest_pep]
out_longest_pep_name = species1.protein.fa, species2.protein.fa, species3.protein.fa, species4.protein.fa
thread = 4
quota_Anchor pre_col -c pre_collinearity.conf -only_longest_pep