process_align

Purpose: Alignment of raw reads to reference genome

Input:

• Experiment.csv
• Species
• Experiment Settings.csv
• Paired_End (boolean)
• Has_Strand_Specific_Information (boolean)
• Used_Spike_Ins (boolean)
• Trimmed *.fastq.gz

Reference: Human = GRCh38p12 Mouse = GRCm38p6 Spike-in = ERCC92 (if spike ins used)

Outputs for later processes: *.bam (sorted) *.alignment.summary.txt *.bai

Process:

• Single End
  • -x: Reference Index
  • -U: fastq file
  • --rna-strandness: R or F or remove
• Paired End
  • -x: Reference Index
  • -1: fastq read 1 file
  • -2: fastq read 1 file
  • --rna-strandness: R or F or remove
  • --no-mixed: Only align paired reads
  • --no-discordant: Only align properly paired reads
• Samtools: sam to bam
  • samtools view -bS -F 4 -F 8 -F 256
• Samtools: index
  • samtools index

Tools: HISAT2 Samtools