T. pratense based mapping

# QC
# The following command was run once for each sample
fastqc AACHLGWM5-7931-01-49-01_S1_L001_R1_001.fastq AACHLGWM5-7931-01-49-01_S1_L001_R2_001.fastq -o /Volumes/archive/userdata/student_users/olivernewman/2023/Illumina_downloads_2023/fastQC/

# MultiQC
multiqc /Volumes/archive/userdata/student_users/olivernewman/2023/Illumina_downloads_2023/fastQC_10_samples_2023/

# Trimming and pairing fastq files
# The following command was run once for each sample
java -jar /usr/local/bin/trimmomatic.jar PE /Volumes/archive/userdata/student_users/olivernewman/2023/Illumina_downloads_2023/OG7931-641472834_fastqs/AACHLGWM5-7931-01-49-01_L1-ds.e02e8eb6bd8a439c90d33e767a56659e/AACHLGWM5-7931-01-49-01_S1_L001_R1_001.fastq /Volumes/archive/userdata/student_users/olivernewman/2023/Illumina_downloads_2023/OG7931-641472834_fastqs/AACHLGWM5-7931-01-49-01_L1-ds.e02e8eb6bd8a439c90d33e767a56659e/AACHLGWM5-7931-01-49-01_S1_L001_R2_001.fastq Sample_1_R1_Paired_trimmed.fastq Sample_1_R1_Unpaired_trimmed.fastq Sample_1_R2_Paired_trimmed.fastq Sample_1_R2_Unpaired_trimmed.fastq ILLUMINACLIP:adapters.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36


# Indexing T. reference genome
STAR --runMode genomeGenerate --genomeDir STAR_ref_index_T_pratense/ --genomeFastaFiles data/GCF_020283565.1_RefSeq/GCF_020283565.1_ARS_RC_1.1_T_pratense_ref_genome.fa --sjdbGTFfile data/GCF_020283565.1_RefSeq/RefSeq_T_pratense.gtf --genomeSAindexNbases 13 --runThreadN 24

# Aligning paired-trimmed fastq files to indexed reference genome
# The following command was run once for each sample
STAR --runMode alignReads --genomeDir /Volumes/userdata/student_users/olivernewman/2023/Reference_genome/ncbi_dataset_Trifolium_pratense_reference_genome/STAR_ref_index_T_pratense/ --outSAMtype BAM SortedByCoordinate --readFilesIn Paired_and_Unpaired_trimmed/Sample_10_R1_Paired_trimmed.fastq Paired_and_Unpaired_trimmed/Sample_10_R2_Paired_trimmed.fastq --runThreadN 72 --outFileNamePrefix /Volumes/userdata/student_users/olivernewman/2023/Illumina_downloads_2023/Mapped_to_T_pratense_genome/ --outFilterMultimapNmax 10


# Counting reads per annotated genes in reference genome using BAM files generated from the previous step
featureCounts -a Reference_genomes/ncbi_dataset_Trifolium_pratense_reference_genome/data/GCF_020283565.1_RefSeq/RefSeq_T_pratense.gtf -o FeatureCounts_10_samples_T_pratense_counts.txt -T 64 Illumina_downloads_2023/Mapped_to_T_pratense_genome/*.bam -p -M --fraction

# Sorting BAM files 
samtools sort Sample_1_Aligned.sortedByCoord.out.bam

# Indexing and sorting T. pratense .gtf file
samtools index RefSeq_T_pratense.gtf
samtools sort RefSeq_T_pratense.gtf