4_alignment_bowtie2.txt

#warming_AK

#Adriana L. Romero-Olivares' protocol for processing RNAseq data of soil 
#Section 4 
#File name: 4_align_bowtie2
#Step: Alignment of reads with bowtie2 

#this protocol uses bowtie2 as a tool to align reads to the reference meta-transcriptome assembled with Trinity

#first build an index for the reference meta-transcriptome 

module load bowtie2/2.2.7
module load samtools/1.3

bowtie2-build Trinity_ALL_f.Trinity.fasta Trinity_ALL_f.Trinity_output.fasta
  
#next, align reads from control and treatment on separate processes
#here, I ran two different alignments, one for control samples and one for treatment samples
#you will need paired and unpaired files generated with Trimommatic on step 1 

#for control 

module load bowtie2/2.2.7
module load samtools/1.3

bowtie2 --local --rf -p 45 -x Trinity_ALL_f.Trinity_output.fasta -1 file_trimmed.r1_paired.fq -2 file_trimmed.r2_paired.fq -U file_trimmed.r1_unpaired.fq,file_trimmed.r2_unpaired.fq -S bowtie2.CALL_aligned_rf.sam

#for treatment 

module load bowtie2/2.2.7
module load samtools/1.3

bowtie2 --local --rf -p 45 -x Trinity_ALL_f.Trinity_output.fasta -1 file_trimmed.r1_paired.fq -2 file_trimmed.r2_paired.fq -U file_trimmed.r1_unpaired.fq,file_trimmed.r2_unpaired.fq -S bowtie2.WALL_aligned_rf.sam

#convert files from sam to bam 

module load samtools/1.3

samtools view -bS -@5 bowtie2.CALL_aligned_rf.sam > bowtie2.CALL_aligned_rf.bam
samtools view -bS -@5 bowtie2.WALL_aligned_rf.sam > bowtie2.WALL_aligned_rf.bam

#sort bam files 

module load samtools/1.3

samtools sort -@20 -n -o bowtie2.CALL_sorted_rf.bam bowtie2.CALL_aligned_rf.bam
samtools sort -@20 -n -o bowtie2.WALL_sorted_rf.bam bowtie2.WALL_aligned_rf.bam

#files _sorted_rf.bam are used for downstream analyses