Pre‐meet commands

1 Set the genome index environment variable

cd /home/train/Pre_meet

export genome_index=Example_output/Hisat2/db/Athaliana_447_TAIR10_Chr3_clean

2 Perform Hisat2 alignment

2.1 Sample SRR5956444 - redirect alignment output, sort, convert to bam format and index the bam

rm -rf Hisat2 Stringtie && mkdir -p Hisat2/SRR5956444 && hisat2 --max-intronlen 50000  --rna-strandness RF -p 2 -x $genome_index -1 Inputs/Reads/SRR5956444/*R1.*fastq.gz -2 Inputs/Reads/SRR5956444/*R2.*fastq.gz | samtools sort -@ 2 -o Hisat2/SRR5956444/Hisat2.bam && samtools index Hisat2/SRR5956444/Hisat2.bam

2.2 Align all samples

for folder in Inputs/Reads/SR*; do echo -e "\n\n## Running Hisat2 on sample ${folder} ..." && folder=$(basename $folder) && mkdir -p Hisat2/${folder} && hisat2 --max-intronlen 50000  --rna-strandness RF -p 2 -x $genome_index -1 Inputs/Reads/${folder}/*R1.*fastq.gz -2 Inputs/Reads/${folder}/*R2.*fastq.gz | samtools sort -@ 2 -o Hisat2/${folder}/Hisat2.bam && samtools index Hisat2/${folder}/Hisat2.bam; done

3 Transcript assemblies

3.1 Assemble all samples using Stringtie

for folder in Inputs/Reads/SRR*; do echo -ne "\n\n## Running stringtie on sample ${folder} ..." && folder=$(basename ${folder}) && mkdir -p Stringtie/${folder} && stringtie Hisat2/${folder}/Hisat2.bam -l ${folder}_STRG -o Stringtie/${folder}/${folder}.gtf > Stringtie/${folder}/stringtie.log 2>&1 && echo done; done

4 Check assemblies for any differences

4.1 Check Stringtie samples

for folder in Inputs/Reads/SRR*; do echo -ne "\n\n## Check stringtie output on sample ${folder} ... Mismatch = " && folder=$(basename ${folder}) && diff Example_output/Stringtie/${folder}/${folder}.gtf Stringtie/${folder}/${folder}.gtf | wc -l && echo done; done

Expected output:

## Check stringtie output on sample Inputs/Reads/SRR5956440 ... Mismatch = 0
done


## Check stringtie output on sample Inputs/Reads/SRR5956444 ... Mismatch = 0
done