scripts/STAR_commands_ReadsToAs2.txt
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#P. citri all data (VF+MF susphire and Edinburgh all Pcitri samples)

ulimit -n 32000

##mapping reads back to rnaSPAdes assembly using STAR to check the percentage of reads that map

cd /DATA/markop/Pcitri_rnaspades_alldata
mkdir star_index

#the soft filtered file was used as a reference
STAR --runThreadN 32 --runMode genomeGenerate --genomeDir ./star_index --genomeFastaFiles ./output/soft_filtered_transcripts.fasta --limitGenomeGenerateRAM=220000000000


mkdir STAR_mapToAssembly

#this command doesn't work because you cannot have paired and single end reads in same mapping!!!
#also with multiple PE samples separated with comma I get a Segmentation fault error :( will do each sample separately
# STAR \
# --runMode alignReads \
# --runThreadN 32 \
# --genomeDir ./star_index \
# --readFilesCommand gzip -c \
# --readFilesIn \
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S1_VF_2_GGAAGAGA-CGAGAGAA_L007_R1_trimmed_paired.R1.fastq.gz \
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S1_VF_2_GGAAGAGA-CGAGAGAA_L007_R1_trimmed_paired.R2.fastq.gz,\
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S1_VF_2_GGAAGAGA-CGAGAGAA_L007_R1_trimmed_orphans.R1.fastq.gz,\
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S2_VF_3_TCGGATTC-CGCAACTA_L007_R1_trimmed_paired.R1.fastq.gz \
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S2_VF_3_TCGGATTC-CGCAACTA_L007_R1_trimmed_paired.R2.fastq.gz,\
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S2_VF_3_TCGGATTC-CGCAACTA_L007_R1_trimmed_orphans.R1.fastq.gz,\
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S3_VF_5_CTGTACCA-CACAGACT_L007_R1_trimmed_paired.R1.fastq.gz \
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S3_VF_5_CTGTACCA-CACAGACT_L007_R1_trimmed_paired.R2.fastq.gz,\
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S3_VF_5_CTGTACCA-CACAGACT_L007_R1_trimmed_orphans.R1.fastq.gz,\
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S4_VF_6_GAGAGTAC-TGGAAGCA_L007_R1_trimmed_paired.R1.fastq.gz \
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S4_VF_6_GAGAGTAC-TGGAAGCA_L007_R1_trimmed_paired.R2.fastq.gz,\
# /FITO_ws/markop/pcitri_trimmed/PRO1976_S4_VF_6_GAGAGTAC-TGGAAGCA_L007_R1_trimmed_orphans.R1.fastq.gz \
# --outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR \
# --outSAMtype BAM SortedByCoordinate \
# --outReadsUnmapped Fastx


# default --outFilterMultimapNmax 20

##mapping only paired reads

#ulimit default 1024
ulimit -n 10000

mkdir STAR_mapToAssembly


#load genome 
STAR --genomeLoad LoadAndExit --genomeDir ./star_index


#SUSPHIRE_VF1
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/VF_02_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/VF_02_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF02_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#SUSPHIRE_VF2
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/VF_03_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/VF_03_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF03_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#SUSPHIRE_VF3
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/VF_05_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/VF_05_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF05_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#SUSPHIRE_VF4
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/VF_06_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/VF_06_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF06_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 



#SUSPHIRE_MF1
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/MF_08_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/MF_08_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF08_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 
#SUSPHIRE_MF2
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/MF_09_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/MF_09_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF09_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 


#SUSPHIRE_MF3
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/MF_10_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/MF_10_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF10_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#SUSPHIRE_MF4
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/MF_11_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2018_mpe_Pcitri/data/MF_11_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF11_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 
#***

#Edinburgh_ALL1
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_ALL_1_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_ALL_1_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EALL1_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#Edinburgh_F1
STAR \
--runMode alignReads \
--runThreadN 6 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_F_1_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_F_1_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF1_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#Edinburgh_F2
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_F_2_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_F_2_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF2_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#Edinburgh_F3
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_F_3_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_F_3_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF3_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#Edinburgh_M1
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_M_1_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_M_1_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM1_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#Edinburgh_M2
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_M_2_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_M_2_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM2_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 

#Edinburgh_M3
STAR \
--runMode alignReads \
--runThreadN 32 \
--genomeDir ./star_index \
--readFilesIn \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_M_3_1.fastq.gz \
/NFSs-datarepo/ngs_omics/NGS_pci_rna-seq_2019_mpe_SUSPHIRE-Edinburgh/data/PC_M_3_2.fastq.gz \
--outFileNamePrefix ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM3_ \
--outSAMtype BAM SortedByCoordinate \
--readFilesCommand pigz -c -d 


#unload genome
STAR --genomeLoad Remove --genomeDir ./star_index




##### we also want to count the reads that are mapped to each transcript and perform DE analysis in R (limma-voom)

#index bam files

samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EALL1_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF10_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM2_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM1_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF05_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF09_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF06_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF3_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM3_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF02_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF08_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF11_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF2_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF03_Aligned.sortedByCoord.out.bam
samtools index -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF1_Aligned.sortedByCoord.out.bam


#### get counts with samtools idxstats

samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EALL1_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EALL1.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF10_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF10.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM2_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM2.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM1_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM1.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF05_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF05.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF09_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF09.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF06_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF06.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF3_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF3.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM3_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EM3.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF02_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF02.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF08_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF08.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF11_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SMF11.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF2_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF2.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF03_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_SVF03.counts
samtools idxstats -@ 6 ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF1_Aligned.sortedByCoord.out.bam > ./STAR_mapToAssembly/Pcitri_mapToAssembly_STAR_EF1.counts



#run R to combine all counts in to an expression matrix -> did it localy on my disk D:\SUSPHIRE\Pcitri\Pcitri_rnaspades_alldata
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Created: 16th May 2022 at 12:05

Last updated: 6th Nov 2024 at 15:26

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Version 1 (earliest) Created 16th May 2022 at 12:05 by Marko Petek

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