This is a pipeline in progress for detecting viral sequences from RNA-seq data. I’ve based it off methods used in this paper: https://www.nature.com/articles/s41564-024-01796-6

Software:

• Trimmomatic
• SPAdes
• Virsorter2, checkV, DRAMv

Input data:

• RNA-seq data in fastq format

Workflow:

• Trim adapter sequences and low quality reads using trimmomatic
• de novo transcriptome assembly with rnaviralSPAdes
• Follow protocol described here to detect viral sequences in assembly

Steps:

1. Use trimmomatic (downloaded on HPC). For example:

java -jar /programs/trimmomatic/trimmomatic-0.39.jar PE -phred33 input_forward.fastq input_reverse.fastq output_forward_paired.fastq output_forward_unpaired.fastq output_reverse_paired.fastq output_reverse_unpaired.fastq ILLUMINACLIP:/programs/trimmomatic/adapters/TruSeq3-PE-2.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:35

• this command accomplishes the following:
  • Removes adapters (ILLUMINACLIP:TruSeq3-PE.fa:2:30:10)
    • ILLUMINACLIP:<fastaWithAdaptersEtc>:<seed mismatches>:<palindrome clip threshold>:<simple clip threshold>
      • fastaWithAdaptersEtc: specifies the path to a fasta file containing all the adapters, PCR sequences etc. The naming of the various sequences within this file determines how they are used. See below.
      • seedMismatches: specifies the maximum mismatch count which will still allow a full match to be performed
      • palindromeClipThreshold: specifies how accurate the match between the two 'adapter ligated' reads must be for PE palindrome read alignment.
      • simpleClipThreshold: specifies how accurate the match between any adapter etc. sequence must be against a read.
    • note, the path to the adapter file on the HPC is /programs/trimmomatic/adapters/TruSeq3-PE-2.fa
  • Removes leading low quality or N bases (below quality 3) (LEADING:3)
  • Removes trailing low quality or N bases (below quality 3) (TRAILING:3)
  • Scan the read with a 4-base wide sliding window, cutting when the average quality per base drops below 15 (SLIDINGWINDOW:4:15)
  • Drop reads below the 35 bases long (MINLEN:35)