HybPhyloMaker "rapid instructions" (June 2025)
Tomas Fer (tomas.fer@natur.cuni.cz)

1. Log in to skirit (skirit.ics.muni.cz aka brno2) MetaCentrum frontend
ssh metausername@skirit.ics.muni.cz

2. Clone HybPhyloMaker from GitHub and move HybSeqSource folder to home folder, download reference
git clone https://github.com/tomas-fer/HybPhyloMaker
mv HybPhyloMaker/HybSeqSource/ .
cd HybSeqSource
wget https://raw.githubusercontent.com/tomas-fer/HybSeqCourse/master/curcuma_HybSeqProbes_first958.fa
cd ../HybPhyloMaker

3. Copy test dataset (Hlavata et al. 2023. Molecular Phylogenetics and Evolution 178, 107666)
cp -r /storage/projects2/hybseqcourse/Amomum .

OR (in case the command above is not working)
mkdir Amomum
cd Amomum
wget https://botany.natur.cuni.cz/fer/hybseqcourse/10rawreads.zip
unzip 10rawreads
rm 10rawreads.zip
cd ..

4. Edit file settings.cfg (Ctrl+O to save changes, Ctrl+X to exit editor)
nano settings.cfg
location=1
server=brno2
data=HybPhyloMaker/Amomum
OUTGROUP="Zingiber-officinale_S242"
MISSINGPERCENT=75
SPECIESPRESENCE=40
probes=curcuma_HybSeqProbes_first958.fa
raxmlperjob=1
phypartsbs=50

5. Prepare pseudoreference
qsub HybPhyloMaker0b_preparereference.sh

6. Install appropriate R packages (and wait until this and previous jobs are finished)
(HybPhyloMaker0b_preparereference.sh - need to be finished prior to running HybPhyloMaker2a_readmappingParallel.sh)
(HybPhyloMaker0c_Rsetup_MetaCentrum.sh - need to be finished prior to running HybPhyloMaker5_missingdataremoval.sh)
qsub HybPhyloMaker0c_Rsetup_MetaCentrum.sh #(this runs for ca. 45mins)

7. Filter the raw data
qsub HybPhyloMaker1a_rawprocessParallel.sh

8. Run this submitter created by the previous job (this script submits 35 jobs)
cd Amomum/20filtered/ && ./submitRawProcessJobs.sh && cd ../..

9. Run the summary script (after all jobs are finished)
qsub HybPhyloMaker1a2_rawprocessParallel_summary.sh

10. Map reads to pseudoreference
qsub HybPhyloMaker2a_readmappingParallel.sh

11. Run this submitter created by the previous job (this script submits 35 jobs)
cd Amomum/exons/21mapped_bwa/ && ./submitMappingJobs.sh && cd ../../..

12. Run the summary script (after all jobs are finished)
qsub HybPhyloMaker2a2_readmappingParallel_summary.sh

13. Generate PSLX files
qsub HybPhyloMaker3_generatepslx.sh

14. Copy PSLX files
mkdir pslx_Amomum2025
cp Amomum/exons/50pslx/*.pslx pslx_Amomum2025

15. Edit file settings.cfg
nano settings.cfg
otherpslx=HybPhyloMaker/pslx_Amomum2025

16. Generate exon assemblies
qsub HybPhyloMaker4a_processpslx.sh

17. Filter missing data
qsub HybPhyloMaker5_missingdataremoval.sh

18. Generate RAxML trees
qsub HybPhyloMaker6a_RAxML_for_selected.sh

19. Run this submitter created by the previous job (this script submits 246 jobs)
cd Amomum/exons/72trees75_40/RAxML/ && ./submitRAxMLjobs.sh && cd ../../../..

20. Run the summary script (after all jobs are finished)
qsub HybPhyloMaker6a2_RAxML_trees_summary.sh

21. Create tree files
qsub HybPhyloMaker7_roottrees.sh

22. Create diverse species trees (run all four, no need to wait...)
qsub HybPhyloMaker8a_astral.sh
qsub HybPhyloMaker8a2_astral4.sh
qsub HybPhyloMaker8b_astrid.sh
qsub HybPhyloMaker8c_mrl.sh
qsub HybPhyloMaker8e_concatenatedFastTree.sh
qsub HybPhyloMaker8f_concatenatedExaML.sh

23. After all jobs are finished run these additional scripts (run all five, no need to wait...)
qsub HybPhyloMaker8h_neighbourNetwork.sh
qsub HybPhyloMaker8k_quartetsampling.sh
qsub HybPhyloMaker8n_SNPheatmap.sh
qsub HybPhyloMaker11_phyparts.sh
qsub HybPhyloMaker15_combinetrees.sh

24. Make a subselection (19 samples as clade representatives), download excludelist.txt and set folder name
cp /storage/projects2/hybseqcourse/excludelist.txt ../HybPhyloMaker/HybSeqSource
nano settings
selection=forNetwork

25. Run script to filter alignments
qsub HybPhyloMaker12_excludesamples.sh

26. Filter alignments, create gene trees and ASTRAL tree (run one by one)
qsub HybPhyloMaker5_missingdataremoval.sh
qsub HybPhyloMaker6a_RAxML_for_selected.sh
cd Amomum/exons/forNetwork/72trees75_40/RAxML/ && ./submitRAxMLjobs.sh && cd ../../../../..
qsub HybPhyloMaker6a2_RAxML_trees_summary.sh
qsub HybPhyloMaker7_roottrees.sh
qsub HybPhyloMaker8a_astral.sh

27. Install julia packages (necessary for SNaQ)
qsub HybPhyloMaker0d_julia_setup_MetaCentrum.sh

28. Run network analyses (SNaQ takes approx. 3 days, PhyloNet approx. 3-6 hours)
qsub HybPhyloMaker8l_SNaQ.sh
qsub HybPhyloMaker8m_PhyloNet.sh
cd Amomum/exons/forNetwork/72trees75_40/RAxML/species_trees/PhyloNet && ./submitPhyloNetjobs.sh && cd ../../../../../../..
qsub HybPhyloMaker8m2_PhyloNet_summary.sh