Alignment

Tom-Heiz1706 Index Code

Copy the index script from the Tom directory into the Portfolio directory

#make sure you are in the Portfolio directory

cp /share/bitcpt/Fall2022/lscapozi/Tom/Tom.starindex.sh .

Rename the file to reflect the Tom-Heinz1706 data

mv Tom.starindex.sh Hz.starindex.sh

Edit the script

#!/bin/tcsh

#BSUB -J starindices_Hz_CherryTom #job name

#BSUB -n 10 #number of nodes

#BSUB -W 2:0 #time for job to complete

#BSUB -o starindices.out.%J #output file

#BSUB -e starindices.err.%J #error file

# For running star to generate genome index

# Run in working directory /share/bitcpt/Fall2022/lscapozi/Portfolio

# Must run this in working directory with subdirectory named /starindices

module load conda

conda activate /usr/local/usrapps/bitcpt/star

set IN=/share/bitcpt/Fall2022/referenceGenomes/Solanum_lycopersicum/Portfolio/Tom-Heinz1706

STAR --runThreadN 10 --runMode genomeGenerate --genomeSAindexNbases 13 --genomeDir starindices --genomeFastaFiles ${IN}/Tom-Heinz_assembly.fasta --sjdbGTFfile ${IN}/Tom-Heinz.agat.gtf --sjdbOverhang 58

Run the index Tom-Heinz1706 code

bsub <Hz.starindex.sh

Check the output

ll starindices

Figure 1: Heinz 1706 Tomato indexing output

Tom-Heinz1706 Alignment Code

Copy the alignment script from the Tom directory to the Portfolio directory

#make sure you are in the portfolio directory

cp /share/bitcpt/Fall2022/lscapozi/Tom/Tom.staralign.sh .

Rename the file

mv Tom.staralign.sh Hz.staralign.sh

Edit code

vi Hz.staralign.sh

#a new window will open with the following information

#!/bin/tcsh

#BSUB -J Hz_staralign #job name

#BSUB -n 12 #number of threads

#BSUB -W 10:0 #time for job to complete

#BSUB -R span[hosts=1] #to keep tasks on one node

#BSUB -R "rusage[mem=20000]" #to request a node with 20MB of memory

#BSUB -o Tom_staralign_%J.out #output file

#BSUB -e Tom_staralign_%J.err #error file

#to align RNA-seq reads to indexed genome using STAR

#STAR cannot make use of HPC MPI, must have -R options to set 1 node & memory

#set threads under 12 on Henry2

#input of indexed genome path is /share/bitcpt/Fall2022/lscapozi/Portfolio/starindices

#input of sequence reads path is /share/bitcpt/Fall2022/CleanData/Solanum_lycopersicum/

#output of aligned reads will go into STAR_align_Portfolio subdirectory in working directory

module load conda

conda activate /usr/local/usrapps/bitcpt/star

# SET IN VARIABLES

set IN=/share/bitcpt/Fall2022/CleanData/Solanum_lycopersicum

set index=starindices

set out=AlignedToTranscriptome

################################

## Leaf Rep 1

################################

# RNA-seq data are in format Sl_Leaf_Rep1_3X_1.fp.fq.gz

set S=Sl_Leaf_Rep1_3X

set EN=fp.fq.gz

# Print the file name to make sure it is right

echo ${IN}/${S}_1.${EN}

STAR --runThreadN 12 --runMode alignReads --genomeDir ${index} --outFileNamePrefix ${out}/${S}_ --readFilesIn ${IN}/${S}_1.${EN} ${IN}/${S}_2.${EN} --readFilesCommand zcat --outSAMtype BAM Unsorted --twopassMode Basic --quantMode TranscriptomeSAM

################################

## Leaf Rep 2

################################

# RNA-seq data are in format Sl_Leaf_Rep2_3X_1.fp.fq.gz

set S=Sl_Leaf_Rep2_3X

set EN=fp.fq.gz

# Print the file name to make sure it is right

echo ${IN}/${S}_1.${EN}

STAR --runThreadN 12 --runMode alignReads --genomeDir ${index} --outFileNamePrefix ${out}/${S}_ --readFilesIn ${IN}/${S}_1.${EN} ${IN}/${S}_2.${EN} --readFilesCommand zcat --outSAMtype BAM Unsorted --twopassMode Basic --quantMode TranscriptomeSAM

################################

## Leaf Rep 3

################################

# RNA-seq data are in format Sl_Leaf_Rep3_3X_1.fp.fq.gz

set S=Sl_Leaf_Rep3_3X

set EN=fp.fq.gz

# Print the file name to make sure it is right

echo ${IN}/${S}_1.${EN}

STAR --runThreadN 12 --runMode alignReads --genomeDir ${index} --outFileNamePrefix ${out}/${S}_ --readFilesIn ${IN}/${S}_1.${EN} ${IN}/${S}_2.${EN} --readFilesCommand zcat --outSAMtype BAM Unsorted --twopassMode Basic --quantMode TranscriptomeSAM

################################

## SAM Rep 1

################################

#RNA-seq data are in format Sl_SAM_Rep1_3X_1.fp.fq.gz

set S=Sl_SAM_Rep1_3X

set EN=fp.fq.gz

#Print the file name to make sure it is right

echo ${IN}/${S}_1.${EN}

STAR --runThreadN 12 --runMode alignReads --genomeDir ${index} --outFileNamePrefix ${out}/${S}_ --readFilesIn ${IN}/${S}_1.${EN} ${IN}/${S}_2.${EN} --readFilesCommand zcat --outSAMtype BAM Unsorted --twopassMode Basic --quantMode TranscriptomeSAM

################################

## SAM Rep 2

################################

#RNA-seq data are in format Sl_SAM_Rep2_3X_1.fp.fq.gz

set S=Sl_SAM_Rep2_3X

set EN=fp.fq.gz

#Print the file name to make sure it is right

echo ${IN}/${S}_1.${EN}

STAR --runThreadN 12 --runMode alignReads --genomeDir ${index} --outFileNamePrefix ${out}/${S}_ --readFilesIn ${IN}/${S}_1.${EN} ${IN}/${S}_2.${EN} --readFilesCommand zcat --outSAMtype BAM Unsorted --twopassMode Basic --quantMode TranscriptomeSAM

################################

## SAM Rep 3

################################

#RNA-seq data are in format Sl_SAM_Rep3_3X_1.fp.fq.gz

set S=Sl_SAM_Rep3_3X

set EN=fp.fq.gz

#Print the file name to make sure it is right

echo ${IN}/${S}_1.${EN}

STAR --runThreadN 12 --runMode alignReads --genomeDir ${index} --outFileNamePrefix ${out}/${S}_ --readFilesIn ${IN}/${S}_1.${EN} ${IN}/${S}_2.${EN} --readFilesCommand zcat --outSAMtype BAM Unsorted --twopassMode Basic --quantMode TranscriptomeSAM

################################

## SAM Rep 4

################################

#RNA-seq data are in format Sl_SAM_Rep4_3X_1.fp.fq.gz

set S=Sl_SAM_Rep4_3X

set EN=fp.fq.gz

#Print the file name to make sure it is right

echo ${IN}/${S}_1.${EN}

STAR --runThreadN 12 --runMode alignReads --genomeDir ${index} --outFileNamePrefix ${out}/${S}_ --readFilesIn ${IN}/${S}_1.${EN} ${IN}/${S}_2.${EN} --readFilesCommand zcat --outSAMtype BAM Unsorted --twopassMode Basic --quantMode TranscriptomeSAM

Run the Alignment job

bsub <Hz.staralign.sh