4.5 Quality control overview

The quality-control module checks aligned RNA-seq and Ribo-seq data before downstream gene-level, codon-level, smORF, and SeRP analyses.

This module covers BAM inspection, enzymatic digestion bias, offset estimation, density generation, density merging, periodicity, metagene profiles, coverage, and sample correlation.

Recommended workflow

rpf_Check
  ↓
rpf_Digest
  ↓
rna_Offset / rpf_Offset
  ↓
rna_Density / rpf_Density
  ↓
merge_dst_list → rpf_Merge
  ↓
rpf_Periodicity / rpf_Metaplot / rpf_Coverage / rpf_Percent / rpf_Corr

Module summary

Section	Main command	Purpose
4.5.1 Check	rpf_Check	Inspect aligned BAM files, filter transcriptome reads, calculate read-length distribution, and optionally evaluate saturation.
4.5.2 Enzymatic bias	rpf_Digest	Detect 5' and 3' end sequence preferences caused by nuclease digestion, ligation, or library-preparation bias.
4.5.3 Offset table	rna_Offset, rpf_Offset	Generate RNA-seq offset tables and predict Ribo-seq P-site offset tables.
4.5.4 Read density	rna_Density, rpf_Density	Convert BAM alignments to transcript-level density files.
4.5.5 Merge density	merge_dst_list, rpf_Merge	Build density file lists and merge single-sample density files into multi-sample matrices.
4.5.6 Periodicity	rpf_Periodicity	Calculate frame-specific read distribution and 3-nt periodicity.
4.5.7 Metaplot	rpf_Metaplot	Generate TIS/TTS-centered metagene profiles.
4.5.8 Coverage	rpf_Coverage, rpf_Percent	Evaluate read coverage across transcript regions and summarize coverage percentage.
4.5.9 Correlation	rpf_Corr	Calculate sample correlation at gene level and nucleotide-position level.

Suggested directory structure

mkdir -p 01.check 02.digestion 03.offset 04.density 05.merge \
         06.periodicity 07.metaplot 08.coverage 09.correlation

Input dependency

Step	Main input	Main output
Check	transcriptome BAM and gene.norm.txt	filtered BAM and read-length statistics
Digestion bias	filtered BAM, transcript annotation, transcript FASTA	end-sequence motif and digestion-site profiles
Offset table	filtered BAM or read-length range	RNA/Ribo offset tables
Density	filtered BAM, offset table, transcript annotation, transcript FASTA	single-sample density file
Merge density	single-sample density files	merged density matrix
Downstream QC	merged density matrix	periodicity, metaplot, coverage, and correlation summaries

Notes

• Run rpf_Check before density generation so that downstream steps use filtered and indexed BAM files.
• Use rna_Offset for RNA-seq and rpf_Offset for Ribo-seq.
• Ribo-seq codon-level analysis should only be performed after checking periodicity, metagene pattern, coverage, and replicate correlation.
• Merge helper commands are documented in the page where their outputs are most commonly used.