4.1 Reference preparation¶

Purpose¶

Reference preparation builds all files needed for read classification, alignment, quantification, RPF density construction, and codon-level Ribo-seq analysis.

Inputs¶

Input	Description
genome FASTA	genomic sequence
GTF/GFF3 annotation	gene, transcript, exon, CDS, and UTR annotation
cDNA/transcript FASTA	transcript sequence for rRNA/tRNA/ncRNA/mRNA extraction
feature table	optional NCBI feature table
external tools	Bowtie, STAR, RSEM, gffread

Create directories¶

mkdir -p ./sce/1.reference/
cd ./sce/1.reference/

mkdir cdna genome gtf mrna ncrna rrna trna norm rsem-index star-index

Download reference files¶

wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_genomic.fna.gz
wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_genomic.gtf.gz
wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_genomic.gff.gz
wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_rna.fna.gz
wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/146/045/GCF_000146045.2_R64/GCF_000146045.2_R64_feature_table.txt.gz

gunzip *.gz

Generate cDNA from genome and GFF3¶

gffread \
  -g GCF_000146045.2_R64_genomic.fna \
  GCF_000146045.2_R64_genomic.gff \
  -F \
  -w cdna.fa

Build Bowtie genome index¶

bowtie-build \
  ../GCF_000146045.2_R64_genomic.fna \
  ./genome/genome \
  --threads 12 \
  &>> ./genome/genome_build.log

Build mRNA index¶

grep -i 'gbkey=mRNA' ./cdna.fa | cut -d ' ' -f 1 | cut -c 2- > ./mrna/mrna.ids

retrieve_seq \
  -i ./cdna.fa \
  -n ./mrna/mrna.ids \
  -o ./mrna/mrna.fa \
  &>> ./mrna/mrna_build.log

bowtie-build ./mrna/mrna.fa ./mrna/mrna --threads 12 &>> ./mrna/mrna_build.log

Build rRNA index¶

grep -i 'gbkey=rRNA' ./cdna.fa | cut -d ' ' -f 1 | cut -c 2- > ./rrna/rrna.ids

retrieve_seq \
  -i ./cdna.fa \
  -n ./rrna/rrna.ids \
  -o ./rrna/rrna.fa \
  &>> ./rrna/rrna_build.log

bowtie-build ./rrna/rrna.fa ./rrna/rrna --threads 12 &>> ./rrna/rrna_build.log

Build tRNA index¶

grep -i 'gbkey=tRNA' ./cdna.fa | cut -d ' ' -f 1 | cut -c 2- > ./trna/trna.ids

retrieve_seq \
  -i ./cdna.fa \
  -n ./trna/trna.ids \
  -o ./trna/trna.fa \
  &>> ./trna/trna_build.log

bowtie-build ./trna/trna.fa ./trna/trna --threads 12 &>> ./trna/trna_build.log

Build ncRNA index¶

grep -iE 'gbkey=ncRNA|gbkey=lnc_RNA|gbkey=miRNA|gbkey=snoRNA|gbkey=snRNA|gbkey=misc_RNA' ./cdna.fa \
  | cut -d ' ' -f 1 \
  | cut -c 2- \
  > ./ncrna/ncrna.ids

retrieve_seq \
  -i ./cdna.fa \
  -n ./ncrna/ncrna.ids \
  -o ./ncrna/ncrna.fa \
  &>> ./ncrna/ncrna_build.log

bowtie-build ./ncrna/ncrna.fa ./ncrna/ncrna --threads 12 &>> ./ncrna/ncrna_build.log

`rpf_Reference` full help¶

Function¶

Normalize annotation into RiboParser-compatible transcript tables and sequences.

Command¶

rpf_Reference \
  -g ../GCF_000146045.2_R64_genomic.fna \
  -t ../GCF_000146045.2_R64_genomic.gff \
  -u 30 \
  -o ./norm/gene \
  &>> ./norm/norm_build.log

Required parameters¶

Parameter	Meaning
`-g`, `--genome`	genome FASTA file
`-t`, `--gtf`	GTF or GFF3 annotation file
`-o`, `--output`	output prefix

Optional parameters¶

Parameter	Meaning
`-u`, `--utr`	add pseudo UTR length for leaderless transcripts
`-c`	retain only protein-coding transcripts
`-l`	retain only the longest protein-coding transcript per gene
`-w`	output full message table

Outputs¶

File	Description
`gene.norm.gtf`	normalized GTF
`gene.norm.txt`	RiboParser transcript annotation table
`gene.norm.rna.fa`	normalized transcript FASTA
`gene.norm.cds.fa`	normalized CDS FASTA

Build STAR index¶

STAR \
  --genomeSAindexNbases 11 \
  --runThreadN 12 \
  --runMode genomeGenerate \
  --genomeDir ./star-index \
  --genomeFastaFiles GCF_000146045.2_R64_genomic.fna \
  --sjdbGTFfile ./norm/gene.norm.gtf

Build RSEM index¶

rsem-prepare-reference \
  -p 12 \
  --gtf ../norm/gene.norm.gtf \
  ../GCF_000146045.2_R64_genomic.fna \
  ./rsem-index/rsem

Quality checklist¶

Bowtie indexes exist for genome, mRNA, rRNA, tRNA, and ncRNA.
STAR index directory is complete.
RSEM index files are complete.
gene.norm.txt, gene.norm.rna.fa, and gene.norm.cds.fa are generated.
Chromosome names are consistent across genome, GTF/GFF3, BAM, and normalized reference.