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igseq's Introduction

igseq

igseq is an R Shiny application designed for antibody sequence analysis, physicochemical property analysis, and post-translational modification (PTM) analysis.

Installation

You can install igseq directly from GitHub using the devtools package.

if (!require('devtools')) install.packages('devtools')
devtools::install_github("pengjiao/igseq")

Prerequisites

1. R packages

Before running the igseq application, ensure all necessary R packages are installed and any required configurations are set up. Here is a list of required packages:

  • shiny
  • reticulate
  • DT
  • Peptides
  • stringr
  • tableHTML
  • seqinr

2. Python Configuration and Requirements

The igseq application requires Python for certain functionalities. Ensure Python is installed and configure the necessary Python packages.

For managing the Python environment and dependencies, we recommend using Conda, an open-source package management system and environment management system. It simplifies package management and deployment for Python packages.

Installing Conda

If Conda is not already installed, download and install Miniconda, a minimal installer for Conda. Miniconda is a smaller alternative to Anaconda, suitable for users who wish to minimize disk space usage while leveraging the powerful Conda environment and package manager.

Installing Required Python Packages

The application requires the following Python packages:

  • BioPython
  • pandas
  • abnumber
conda activate
which python # pythonPath used in launchApp function
conda install -c conda-forge biopython pandas
conda install -c bioconda abnumber

3. igblast

The package relies on igblast and requires specifying the igblast path during runtime. Follow these steps for installation and configuration:

  1. The latest igblast program can be downloaded from: NCBI igblast download link
VERSION=1.22.0
wget https://ftp.ncbi.nih.gov/blast/executables/igblast/release/LATEST/ncbi-igblast-${VERSION}-x64-linux.tar.gz
  1. Extract igblast
tar -zxvf ncbi-igblast-${VERSION}-x64-linux.tar.gz
sudo cp ncbi-igblast-${VERSION}/bin/* /bin
  1. Download the reference database
OUTDIR=~/share/igblast
if [ ! -d $OUTDIR ]; then
    mkdir -p $OUTDIR
fi
# Fetch database
wget -q -r -nH --cut-dirs=5 --no-parent     ftp://ftp.ncbi.nih.gov/blast/executables/igblast/release/database     -P ${OUTDIR}/database
# Extract
tar -C ${OUTDIR}/database -xf ${OUTDIR}/database/mouse_gl_VDJ.tar
tar -C ${OUTDIR}/database -xf ${OUTDIR}/database/rhesus_monkey_VJ.tar
cp -r ncbi-igblast-${VERSION}/internal_data $OUTDIR
cp -r ncbi-igblast-${VERSION}/optional_file $OUTDIR
  1. Build igblast database from IMGT reference sequences. This script is modified from fetch_imgtdb.sh of the immcantation workflow, with the addition of downloading IGHJ amino acid.
OUTDIR_IMGT=~/share/germlines/imgt
REPERTOIRE="imgt"
if [ ! -d $OUTDIR_IMGT ]; then
  mkdir -p $OUTDIR_IMGT
fi
SPECIES_QUERY=("human:Homo+sapiens"
               "mouse:Mus"
               "rat:Rattus+norvegicus"
               "rabbit:Oryctolagus+cuniculus"
               "rhesus_monkey:Macaca+mulatta")
COUNT=0
for SPECIES in ${SPECIES_QUERY[@]}
do
    KEY=${SPECIES%%:*}
    VALUE=${SPECIES#*:}
    #REPLACE_VALUE=${SPECIES_REPLACE[$COUNT]#*:}
	echo "Downloading ${KEY} repertoires into ${OUTDIR_IMGT}"
	
	# Download VDJ
	echo "|- VDJ regions"
    FILE_PATH="${OUTDIR_IMGT}/${KEY}/vdj"
    FILE_PATH_AA="${OUTDIR_IMGT}/${KEY}/vdj_aa"
    FILE_PATH_LV="${OUTDIR_IMGT}/${KEY}/leader_vexon"
    mkdir -p $FILE_PATH $FILE_PATH_AA $FILE_PATH_LV
    
    # VDJ Ig
    echo "|---- Ig"
    for CHAIN in IGHV IGHD IGHJ IGKV IGKJ IGLV IGLJ
    do
        URL="https://www.imgt.org/genedb/GENElect?query=7.1+${CHAIN}&species=${VALUE}"
        FILE_NAME="${FILE_PATH}/${REPERTOIRE}_${KEY}_${CHAIN}.fasta"
        TMP_FILE="${FILE_NAME}.tmp"
        #echo $URL
        wget $URL -O $TMP_FILE -q
        awk '/<pre>/{i++}/<\/pre>/{j++}{if(j==2){exit}}{if(i==2 && j==1 && $0!~"^<pre>"){print}}' $TMP_FILE > $FILE_NAME
        # Make sed command work also for mac, see: https://stackoverflow.com/a/44864004
        sed -i.bak "$REPLACE_VALUE" $FILE_NAME && rm $FILE_NAME.bak
        rm $TMP_FILE
    done
    
    # Artificial spliced leader and V exon for Ig
    for CHAIN in IGHV IGKV IGLV
    do
        URL="https://www.imgt.org/genedb/GENElect?query=8.1+${CHAIN}&species=${VALUE}&IMGTlabel=L-PART1+V-EXON"
        FILE_NAME="${FILE_PATH_LV}/${REPERTOIRE}_lv_${KEY}_${CHAIN}.fasta"
        TMP_FILE="${FILE_NAME}.tmp"
        #echo $URL
        wget $URL -O $TMP_FILE -q
        awk '/<pre>/{i++}/<\/pre>/{j++}{if(j==2){exit}}{if(i==2 && j==1 && $0!~"^<pre>"){print}}' $TMP_FILE > $FILE_NAME
        # Make sed command work also for mac, see: https://stackoverflow.com/a/44864004
        sed -i.bak "$REPLACE_VALUE" $FILE_NAME && rm $FILE_NAME.bak
        rm $TMP_FILE
    done
        
    # V and J amino acid for Ig
    for CHAIN in IGHV IGHJ IGKV IGLV
    do
        URL="https://www.imgt.org/genedb/GENElect?query=7.3+${CHAIN}&species=${VALUE}"
        FILE_NAME="${FILE_PATH_AA}/${REPERTOIRE}_aa_${KEY}_${CHAIN}.fasta"
        TMP_FILE="${FILE_NAME}.tmp"
        #echo $URL
        wget $URL -O $TMP_FILE -q
        awk '/<pre>/{i++}/<\/pre>/{j++}{if(j==2){exit}}{if(i==2 && j==1 && $0!~"^<pre>"){print}}' $TMP_FILE > $FILE_NAME
        # Make sed command work also for mac, see: https://stackoverflow.com/a/44864004
        sed -i.bak "$REPLACE_VALUE" $FILE_NAME && rm $FILE_NAME.bak
        rm $TMP_FILE
    done
    # Download leaders
    echo "|- Spliced leader regions"
    FILE_PATH="${OUTDIR_IMGT}/${KEY}/leader"
    mkdir -p $FILE_PATH

    # Spliced leader Ig
    echo "|---- Ig"
    for CHAIN in IGH IGK IGL
    do
        URL="https://www.imgt.org/genedb/GENElect?query=8.1+${CHAIN}V&species=${VALUE}&IMGTlabel=L-PART1+L-PART2"
        FILE_NAME="${FILE_PATH}/${REPERTOIRE}_${KEY}_${CHAIN}L.fasta"
        TMP_FILE="${FILE_NAME}.tmp"
        #echo $URL
        wget $URL -O $TMP_FILE -q
        awk '/<pre>/{i++}/<\/pre>/{j++}{if(j==2){exit}}{if(i==2 && j==1 && $0!~"^<pre>"){print}}' $TMP_FILE > $FILE_NAME
        sed -i.bak "$REPLACE_VALUE" $FILE_NAME && rm $FILE_NAME.bak
        rm $TMP_FILE
    done
    # Download constant regions
    echo "|- Spliced constant regions"
    FILE_PATH="${OUTDIR_IMGT}/${KEY}/constant/"
    mkdir -p $FILE_PATH

    # Constant Ig
    echo "|---- Ig"
    for CHAIN in IGHC IGKC IGLC
    do
        # IMGT does not have artificially spliced IGKC / IGLC for multiple species
        if [ "$CHAIN" == "IGHC" ]; then
            QUERY=14.1
        else
            QUERY=7.5
        fi

        URL="https://www.imgt.org/genedb/GENElect?query=${QUERY}+${CHAIN}&species=${VALUE}"
        FILE_NAME="${FILE_PATH}/${REPERTOIRE}_${KEY}_${CHAIN}.fasta"
        TMP_FILE="${FILE_NAME}.tmp"
        #echo $URL
        wget $URL -O $TMP_FILE -q
        awk '/<pre>/{i++}/<\/pre>/{j++}{if(j==2){exit}}{if(i==2 && j==1 && $0!~"^<pre>"){print}}' $TMP_FILE > $FILE_NAME
        sed -i.bak "$REPLACE_VALUE" $FILE_NAME && rm $FILE_NAME.bak
        rm $TMP_FILE
    done
    echo ""
    ((COUNT++))
done
  1. Process downloaded IMGT reference for igblast
OUTDIR_FA=${OUTDIR}/fasta
TMPDIR=${OUTDIR}/tmpdir
GERMDIR=$OUTDIR_IMGT
if [ ! -d $OUTDIR_FA ]; then
  mkdir -p $OUTDIR_FA
fi
if [ ! -d $TMPDIR ]; then
  mkdir -p $TMPDIR
fi

for SPECIES_Q in ${SPECIES_QUERY[@]}
do
    SPECIES=${SPECIES_Q%%:*}
    for CHAIN in IG
    do
        # VDJ nucleotides
        for SEGMENT in V D J
        do
	        # VDJ nucleotides
	        F=$(echo imgt_${SPECIES}_${CHAIN}_${SEGMENT}.fasta | tr '[:upper:]' '[:lower:]')
	        cat ${GERMDIR}/${SPECIES}/vdj/imgt_${SPECIES}_${CHAIN}?${SEGMENT}.fasta > ${TMPDIR}/${F}
	    done
		# C nucleotides
	    F=$(echo imgt_${SPECIES}_${CHAIN}_c.fasta | tr '[:upper:]' '[:lower:]')
	    cat ${GERMDIR}/${SPECIES}/constant/imgt_${SPECIES}_${CHAIN}?C.fasta > ${TMPDIR}/${F}
	    # V amino acids
        F=$(echo imgt_aa_${SPECIES}_${CHAIN}_v.fasta | tr '[:upper:]' '[:lower:]')
        cat ${GERMDIR}/${SPECIES}/vdj_aa/imgt_aa_${SPECIES}_${CHAIN}?V.fasta > ${TMPDIR}/${F}
	done
done

# J amino acids
for SPECIES_Q in ${SPECIES_QUERY[@]}
do
	SPECIES=${SPECIES_Q%%:*}
	CHAIN=IG
    F=$(echo imgt_aa_${SPECIES}_${CHAIN}_j.fasta | tr '[:upper:]' '[:lower:]')
    cat ${GERMDIR}/${SPECIES}/vdj_aa/imgt_aa_${SPECIES}_${CHAIN}?J.fasta > ${TMPDIR}/${F}
done


# Parse each created fasta file to create igblast database
cd ${TMPDIR}
NT_FILES=$(ls *.fasta | grep -E "imgt_(human|mouse|rhesus_monkey|rat|rabbit).+\.fasta")
for F in ${NT_FILES}; 
do
	edit_imgt_file.pl ${F} | awk '/^>/{if(seen[$0]++) next} {print}' > ${OUTDIR}/fasta/${F}
	makeblastdb -parse_seqids -dbtype nucl -in ${OUTDIR}/fasta/${F} \
        -out ${OUTDIR}/database/${F%%.*}
done

AA_FILES=$(ls *.fasta | grep -E "imgt_aa_(human|mouse|rhesus_monkey|rat|rabbit).+\.fasta")
for F in ${AA_FILES}; do
	edit_imgt_file.pl ${F} | awk '/^>/{if(seen[$0]++) next} {print}' > ${OUTDIR}/fasta/${F}
	makeblastdb -parse_seqids -dbtype prot -in ${OUTDIR}/fasta/${F} \
        -out ${OUTDIR}/database/${F%%.*}
done
cd -
# Remove temporary fasta files
#cd -; rm -rf $TMPDIR

Running the Application

igseq::launchApp()

Features

  • Sequence Analysis: Users can upload a FASTA file or directly paste FASTA content for sequence analysis. The application offers options to select sequence type (Protein or DNA), species, and whether to remove duplicate sequences.
  • Physicochemical Property Analysis: Users can select to analyze various physicochemical properties such as length, molecular weight, net charge, etc. The results are displayed in a table and can be downloaded.
  • Post-Translational Modification Analysis: The application provides an interface to analyze post-translational modifications. The results highlight specific motifs and modifications in the sequence data.

igseq's People

Contributors

pengjiao avatar

Watchers

avatar

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