docker pull fanyucai1/virus docker tag fanyucai1/virus virus
mkdir -p /ref/
2-1:update or download nextclade(Optional: not yet incorporated into the analysis process.)
rm -rf /ref/nextclade_db/ python3 core/update_nextclade_db.py -d /ref/nextclade_db
2-2:virus genome and index(Optional)
mkdir -p /ref/bowtie2/ # Initialize and build the species genome online python3 core/ref_index.py -o /ref/bowtie2/ # Add an extra reference genome sequence: python3 core/ref_index.py -b primer.bed -o /ref/bowtie2/ -n Chikungunya_virus_D250282 -f CHIKV_ref_D250282.fasta # if no bed file exists: python3 core/ref_index.py -o /ref/bowtie2/ -n Chikungunya_virus_D250282 -f CHIKV_ref_D250282.fasta
The currently available list of reference genomes for viral species includes:
Chikungunya_virus
Dengue_virus_type_1
Dengue_virus_type_2
Dengue_virus_type_3
Dengue_virus_type_4
H10N4
H1N1
H3N2
H5N1
H5N6
H5N8
H6N1
H6N2
H7N9
H9N2
HIV-1
Human_Metapneumovirus
Human_adenovirus_B1
Human_adenovirus_F
Human_adenovirus_type_7
Influenza_B_viruses_Victoria
Marburg_Virus
Measles_virus
Monkeypox_virus
Porcine_reproductive_and_respiratory_syndrome_virus_1
RSV-A
RSV-B
SARS-CoV-2
Yellow_fever_virus
Zika_virus
2-3:nt_viruses
Download NCBI database using BLAST:https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ and nt_viruses
dnf install perl-Archive-Tar dnf install perl-JSON-PP mkdir -p /ref/nt_viruses cd /ref/nt_viruses perl ncbi-blast-2.16.0+/bin/update_blastdb.pl nt_viruses --decompress
If above method fails, you can directly download the corresponding database files from the NCBI BLAST database using wget. (https://ftp.ncbi.nlm.nih.gov/blast/db/)
python3 core/download_NCBI_db.py 22
2-4:vsp database:https://help.idm.illumina.com/dragen-microbial-enrichment-plus/dragen-microbial-enrichment-plus Download file:VSPV2_2-7-0_Panel_Summary.xlsx
mkdir -p /ref/VSP/ cd /ref/VSP/ python3 core/VSP.py ncbi-blast-2.14.1+/bin/blastdbcmd -db /ref/nt_viruses/nt_viruses -entry_batch /ref/VSP/accession.list -outfmt "%f" > /ref/VSP/VSP.fasta bowtie2-build /ref/VSP/VSP.fasta /ref/VSP/VSP.fasta
2-5:combine VSP,RVDB and NCBI virus
mkdir -p /ref/NCBI_Nucleotide_Completeness
cd /ref/NCBI_Nucleotide_Completeness
Download NCBI virus Nucleotide (Nucleotide Completeness),Host(Human) and Accession without version:sequences.acc:https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?SeqType_s=Nucleotide&Completeness_s=complete
Download Current Release Reference Viral DataBase(RVDB):https://rvdb.dbi.udel.edu/previous-release
wget https://rvdb.dbi.udel.edu/download/U-RVDBv30.0.fasta.gz
gunzip U-RVDBv30.0.fasta.gz
grep ">" U-RVDBv30.0.fasta|awk -F"|" {print $3} |awk -F"." {print $1}>RVDB_accession.list
sort RVDB_accession.list sequences.acc | uniq -d | cat VSP_accession.list - | sort -u > final.txt
ncbi-blast-2.14.1+/bin/blastdbcmd -db /ref/nt_viruses/nt_viruses -out virus.fasta -entry_batch final.txt -outfmt "%f"
grep ">" virus.fasta|sort -u|awk -F" " '{print $1}'|awk -F">" '{print $2}' >acc.id
rm -rf virus.fasta final.txt
ncbi-blast-2.14.1+/bin/blastdbcmd -db /ref/nt_viruses/nt_viruses -out virus_completeness_unique.fasta -entry_batch acc.id -outfmt "%f"
ncbi-blast-2.14.1+/bin/makeblastdb -in virus_completeness_unique.fasta -dbtype nucl
2-5:kraken2 database:https://benlangmead.github.io/aws-indexes/k2
mkdir -p /ref/kraken/ cd /ref/kraken/ wget https://genome-idx.s3.amazonaws.com/kraken/k2_pluspf_20250402.tar.gz tar xvzf k2_pluspf_20250402.tar.gz
2-5:Download or build host(default:human) genome bowtie2:https://github.com/BenLangmead/bowtie-majref
mkdir -p /ref/host/human/ cd /ref/host/human/ wget -P /ref/host/human/ https://genome-idx.s3.amazonaws.com/bt/grch38_1kgmaj_snvindels_bt2.zip unzip grch38_1kgmaj_snvindels_bt2.zip mkdir -p /ref/host/mouse wget docker run -v /ref/host/mouse:/ref/ virus sh -c 'export PATH=/opt/conda/bin:$PATH && cd /ref/ && bowtie2-build GRCm39.genome.fa GRCm39.genome.fa'
usage: Virus NGS pipeline.
Email:fanyucai3@gmail.com
[-h] -p1 PE1 [PE1 ...]
[-p2 PE2 [PE2 ...]] -p
PREFIX [PREFIX ...] -o
OUTDIR -c CONFIG -l
{50,75,100,150,200,250,300}
[-s SPECIES]
[-t THREADS]
[-plot PLOT] [-fa FA]
[-e BED] [-r REF]
[-b BOWTIE2]
optional arguments:
-h, --help show this help message and exit
-p1 PE1 [PE1 ...], --pe1 PE1 [PE1 ...]
R1 fastq
-p2 PE2 [PE2 ...], --pe2 PE2 [PE2 ...]
R2 fastq
-p PREFIX [PREFIX ...], --prefix PREFIX [PREFIX ...]
prefix of output
-o OUTDIR, --outdir OUTDIR
diretory of output
-c CONFIG, --config CONFIG
config file
-l {50,75,100,150,200,250,300}, --length {50,75,100,150,200,250,300}
read length
-s SPECIES, --species SPECIES
host species name,default:human
-plot PLOT, --plot PLOT
plot when covered_percent default=60
-fa FA, --fa FA output consensus fasta when covered_percent default=70
Reference/Bowtie2 Index Options:
-e BED, --bed BED bed file(Optional)
-r REF, --ref REF ref fasta(Optional)
-b BOWTIE2, --bowtie2 BOWTIE2
directory contains reference bowtie2 index(Optional)
Command-line example:
python3 pipeline.py -p1 sampleID_R1.fastq.gz -p2 sampleID_R2.fastq.gz -p sampleID -o outdir/ -l 150 -c config.ini
python3 pipeline.py -p1 sampleID_R1.fastq.gz -p2 sampleID_R2.fastq.gz -p sampleID -o outdir/ -l 150 -c config.ini -r ref.fasta -b /ref/bowtie2/Chikungunya_virus/ -e primer.bed
python3 pipeline.py -p1 sampleID_R1.fastq.gz -p2 sampleID_R2.fastq.gz -p sampleID -o outdir/ -l 150 -c config.ini -r ref.fasta -b /ref/bowtie2/Chikungunya_virus/