Example

demo for use KK4D

download demo data from https://doi.org/10.6084/m9.figshare.25060220.v1.

Input file list

• Ath.genome.fa.gz
• Ath.gff3.gz
• IRGSP.cds.fa.gz
• IRGSP.pep.fa.gz
• IRGSPRAP.gff3.gz

From genome file get the protein and CDS sequence files for Arabidopsis

gzip -d Ath.genome.fa.gz 
gzip -d Ath.gff3.gz 
bash genome2cdspep.sh Ath.genome.fa Ath.gff3 Ath .

This will output the file Ath.pep and Ath.cds.

Please remember that each new analysis of a different genome must be in a new folder, otherwise it may cause errors.

example1:Comparative analysis of one genomes. (Arabidopsis thaliana)

mkdir Ath_result
cd Ath_result
mv ../Ath.pep .
mv ../Ath.cds .
mv ../Ath.gff3 .
KK4D.sh all -group 1 -cpu 24 -key ID -type mRNA -sample A.thaliana -abbr Ath -gff3 Ath.gff3 -protein Ath.pep -cds Ath.cds -chrnum 5

24 CPU threads were used above

output file list

Ath.Ath.dotplot.pdf A.thaliana.A.thaliana.sankey.pdf ks_4DTv.bold.jpg Ath_Ath.block.coline.pdf

• Ath_Ath.homolog
• All.Ks_peak.csv
• All.X4dtv_corrected_peak.csv
• Ath_Ath.kaks4DTv.csv

All.Ks_peak.csv

x	y	peak_time	type	peak_type
0.047947329	0.005494129	2.715024274	A.thaliana	Ks
0.906953311	1.082462082	51.35635961	A.thaliana	Ks
2.407495406	0.11673559	136.3247682	A.thaliana	Ks

Combined with the Ks distribution chart drawn above, it can be seen that the line in black font in the table is the correct peak value. The x value in this row means that the peak Ks is 0.906953311, and the following peak_time 51.35635961 indicates that the doubling of the Arabidopsis genome occurred at 51.35635961MYA (millions of years ago).

Ks peak and 4DTv peak file, please note that there may be redundant peaks in it, and users need to judge by themselves which are the final valid peaks. The default evolutionary rate λ is 7E-9. If you need to change the value of λ, you can calculate the time of your species according to the formula T=(Ks_peak/2λ). For example, if you need to change λ to 2.5e-9, then You can use T=0.906953311/(2*(2.5e-9))/(1E6) to get T=181.39066219 MYA.

example2:Comparative analysis of two genomes. (Arabidopsis thaliana and rice IRGSP1.0)

mkdir Ath_rice
cd Ath_rice
mv ../Ath.pep .
mv ../Ath.cds .
mv ../Ath.gff3 .
mv ../IRGSP.cds.fa.gz .
mv ../IRGSP.pep.fa.gz .
mv ../IRGSPRAP.gff3.gz .
KK4D.sh all -wd /pathto/Ath_rice -group 2 -cpu 32 -key ID ID -type mRNA mRNA -sample A.thaliana Oryza.Sativa -abbr Ath Osa -gff3 Ath.gff3 IRGSPRAP.gff3.gz -protein Ath.pep IRGSP.pep.fa.gz -cds Ath.cds IRGSP.cds.fa.gz -chrnum 5 12

output file list

A.trichopoda.Oryza.Sativa.sankey.pdf

Ath.Osa.pdf

Ath_Osa.bar.plot.pdf

Ath_Osa.block.coline.pdf

ks_4DTv.bold.pdf

Ks_new.peak.png

X4dtv_corrected_new.peak.png

Ath_Osa.kaks4DTv.csv

Seq	4dtv_corrected	Ka	Ks	Ka/Ks
AT1G01340-Os06t0527100-01	0.517241379	0.325955	3.27552	0.0995124
AT1G01360-Os06t0526400-00	0.422222222	0.350108	3.14752	0.111233
AT1G01430-Os06t0524400-01	0.482352941	0.47486	2.78177	0.170704
AT1G01440-Os06t0524300-01	0.444444444	0.713729	2.12416	0.336005
AT1G01490-Os03t0126700-01	0.45	0.314753	3.76469	0.0836064
AT1G01490-Os10t0532300-01	0.303030303	0.359687	3.56672	0.100845
...	...	...	...	...

All.Ks_peak.csv

x	y	peak_time	type	peak_type
3.087357756	0.605837178	257.279813	A.trichopoda	Ks

All.X4dtv_corrected_peak.csv

x	y	peak_time	type	peak_type
0.44939236	6.75502253	37.44936336	A.trichopoda	X4dtv_corrected