This example shows how to create reference based assemblies of viral transcriptomes.

1. Trimming transcriptome reads:

   1. fastqc (for example, fastqc -o qc_reports R1.fq.gz R2.fq.gz)
   2. trimmomatic (see viral sequence detection)

2. Download reference genomes (Assembly ids listed on a text file called acc_list.txt), and unzip the output from ncbi

   1. Jingmen tick virus: GCF_000919875.1
   2. Alongshan virus: GCA_027256855.1

   programs/ncbi_datasets/datasets download genome accession --inputfile acc_list.txt --include gff3,genome,gbff
   unzip ncbi_dataset.zip
   

3. Index the reference genomes (repeat for each reference)

   # load hisat2
   source /programs/HISAT2/hisat2.sh
   
   #index reference
   hisat2-build GCA_027256855.1_ASM2725685v1_genomic.fna GCA_027256855.1_index
   

4. Map reads to reference

    hisat2 -p 4 -x /path/to/index -1 /path/to/R1 -2 /path/to/R2
    
    # for example,
    hisat2 -p 4 -x ./references/GCA_027256855.1/GCA_027256855.1_index -1 ../HaeL_reads/CM7_2022_93_R1_paired.fastq -2 ../HaeL_reads/CM7_2022_93_R2_paired.fastq 
    
    #sort
    samtools sort -o alnst.sorted.bam alns.sam
   

5. Assemble transcripts using the aligned reads

   mkdir assembly
   programs/bin/cufflinks/gffread ../../references/GCA_027256855.1/GCA_027256855.1.gff -T -o ../../references/GCA_027256855.1/genomic.gtf
   stringtie mapped_reads/aligned_reads.bam -o stringtie_out/transcripts.gtf -p 4