HybPhyloMaker - working with cpDNA off-target data (June 2025)
Tomas Fer (tomas.fer@natur.cuni.cz)

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A. Mapping to regions (both CDS and non-coding)
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1. Log in to skirit (skirit.ics.muni.cz aka brno2) MetaCentrum frontend
ssh metausername@skirit.ics.muni.cz

2. Copy data (takes approx. 3 mins)
cp -r /storage/projects2/hybseqcourse/AmomumFULL .

3. Edit the settings file (set the chloroplast reference)
(Wurfbainia villosa, NC_044746, https://pmc.ncbi.nlm.nih.gov/articles/PMC7113360/)
nano settings.cfg
data=HybPhyloMaker/AmomumFULL
OUTGROUP="Zingiber-officinale_S242"
cp=yes
cpDNACDS=NC_044746_reference_CDSnonHPM200.fa
cpGBnr=NC_044746
otherpslxcp=HybPhyloMaker/pslx_Amomum_cpDNA

4. Run the script 0f to download and prepare HybPhyloMaker compatible reference
(this saves many references to cp/00reference and to HybSeqSource)
qsub HybPhyloMaker0f_createPlastomeRef.sh

5. Run script to prepare pseudoreference
qsub HybPhyloMaker0b_preparereference.sh

6. Filter the raw data
qsub HybPhyloMaker1a_rawprocessParallel.sh

7. Run this submitter created by the previous job (this script submits 35 jobs and takes approx. 12 mins)
cd AmomumFULL/20filtered/ && ./submitRawProcessJobs.sh && cd ../..

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

9. Map reads to chloroplast pseudoreference
qsub HybPhyloMaker2a_readmappingParallel.sh

10. Run this submitter created by the previous job (this script submits 35 jobs and takes approx. 4 mins)
cd AmomumFULL/cp/21mapped_bwa/ && ./submitMappingJobs.sh && cd ../../..

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

12. Continue as with normal HybPhyloMaker analysis - results are now in the 'cp' folder
qsub HybPhyloMaker3_generatepslx.sh
mkdir pslx_Amomum_cpDNA
cp AmomumFULL/cp/50pslx/*.pslx pslx_Amomum_cpDNA/
qsub HybPhyloMaker5_missingdataremoval.sh
qsub HybPhyloMaker8e_concatenatedFastTree.sh

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

2. Download the full plastome reference (one IR removed)
cp /storage/projects2/hybseqcourse/Wurfbainia-villosa_NC044746.fa ../HybSeqSource

OR (in case the command above is not working)
wget https://botany.natur.cuni.cz/fer/hybseqcourse/Wurfbainia-villosa_NC044746.fa
mv Wurfbainia-villosa_NC044746.fa ../HybSeqSource

3. Edit the settings file (set the chloroplast reference - Wurfbainia villosa)
nano settings.cfg
cp=full
cpDNA=Wurfbainia-villosa_NC044746.fa

4. Map reads to full plastome reference
qsub HybPhyloMaker2a_readmappingParallel.sh

5. Run this submitter created by the previous job (this script submits 35 jobs and takes approx. 4 mins)
cd AmomumFULL/fullplastome/21mapped_bwa/ && ./submitMappingJobs.sh && cd ../../..

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

7. Generate alignment of full plastomes (takes approx. 9 mins)
qsub HybPhyloMaker3a_fullalignment.sh

8. Create of FastTree based on full plastome data
qsub HybPhyloMaker8e_concatenatedFastTree.sh
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