RGB EPP

Reference Genome based Exon Phylogeny Pipeline

License: GPL-2.0-only

Author: Guoyi Zhang

Requirements

External software

• fastp
• spades.py (provided by spades)
• diamond
• bowtie2
• samtools
• bcftools
• exonerate (optional, only for --codon)
• java
• macse (default recognized path: /usr/share/java/macse.jar)
• trimal

Internal software

• sortdiamond (default recognized path: /usr/bin/sortdiamond)
• delstop (default recognized path: /usr/bin/delstop)

Arguments

Details

    -c	--config	config file for software path (optional)
    -g	--genes		gene file path (optional, if -r is specified)
    -f	--functions	functions type (optional): all clean assembly 
      	           	 map postmap varcall consen codon align trim
    -h	--help		show this information
    -l	--list		list file path
    -m	--memory	memory settings (optional, default 16 GB)
    -r	--reference	reference genome path
    -t	--threads	threads setting (optional, default 8 threads)
    --codon		Only use the codon region (optional)
    --fastp		Fastp path (optional)
    --spades		Spades python path (optional)
    --diamond		Diamond python path (optional)
    --sortdiamond	SortDiamond python path (optional)
    --bowtie2		Bowtie2 path (optional)
    --samtools		Samtools path (optional)
    --bcftools		Bcftools path (optional)
    --exonerate		Exonerate path (optional)
    --macse		Macse jarfile path (optional)
    --delstop		Delstop path (optional)
    --trimal		Trimal path (optional)
    for example: ./RGBEPP -f all -l list -t 8 -r reference.fasta 

Directories Design

.
├── 00_raw
├── 01_fastp
├── 02_spades
├── 03_bowtie2
├── 04_bam
├── 05_vcf
├── 06_consen
├── 07_macse
├── 08_trimal
├── list
├── gene
├── reference.aa.fasta
└── RGBEPP

Each directory corresponds to each function.

00_raw should conatin all raw fastq.gz data.

Text Files

list is the text file containing all samples, if your raw data is following the style ${list_name}_R1.fastq.gz and ${list_name}_R2.fastq.gz, ${list_name} is what you should list in list file. The easy way to get it in Linux/Unix system is the following command

cd 00_raw
ls | sed "s@_R[12].fastq.gz@@g" | uniq > ../list
cd ..

genes is the text file containing all gene names from the reference fasta file. The easy way to get it in Linux/Unix system is the following command

grep '>' Reference.fasta | sed "s@>@@g" > genes

reference.aa.fasta can be replaced by another other name, but it must contain reference amino acids genome in fasta format

Process

RGBEPP functions

• Function clean: Quality control + trimming (fastp)
• Function assembly: de novo assembly (spades)
• Function map: local nucleic acids alignment search against amino acids subject sequence (diamond, sortdiamond), mapping raw reads to its scaffolds sequences (bowtie2)
• Function postmap: Sorting and marking the read read alignment (samtools)
• Function varcall: variant calling and filtering (bcftools)
• Function consen: get consensus fasta file from vcf files (bcftools), then sort sequences based on gene name and taxa name (RGBEPP)
• Function codon (optional): only extract the exon sequence (exonerate)
• Function align: multiple sequence align based on condon (macse)
• Function trim: trimming based on codon (trimal, delstop)

Arguments reuqirements for functions

Functions	-g/--gene	-l/--list	-r/--reference
clean		✔
assembly		✔
map		✔	✔
postmap		✔
varcall		✔
consen	✔	✔
codon	✔		✔
align	✔
trim	✔

Downstream process

• concatenate sequences via SeqCombGo or catsequences or sequencematrix
• coalescent / concatenated phylogeny

Inner software

sortdiamond

Usage: sortdiamond diamond_output.m8 generated.fasta sseq,qstart,qend,bitscore/evalue,qseq(optional, default 1,6,7,11,17, start from 0) bitscore/evalue(optional, default bitscore)

Default sseq is column 2, qstart is column 8, etc.

Diamond default output format (--outfmt 6) does not contain qseq, you must custom the output format under output format 6.

delstop

delstop <fasta_aa> <fasta_nt> --delete

Delete StopCondon generated by Macse. fasta_aa and fasta_nt should be macse output files, --delete should be used when downstream software is tirmal

splitfasta

Usage: splitfasta sample.fasta

It always creates directories in the path that you run the splitfasta, and puts split fasta into the directory.