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Strainphlan 4.0 analysis Lifelines NEXT

Adapted from Biobakery (StrainPhlAn 4.0). https://github.com/biobakery/MetaPhlAn/wiki/StrainPhlAn-4

Authors: Trishla Sinha, Ranko Gascesa, Description: The script shows how strain profiling was performed using Strainphlan 4 for all maternal and infant samples post QC, for all species.
Languages: Bash and R.

Step 1: Reconstruct all species strains

https://github.com/GRONINGEN-MICROBIOME-CENTRE/gmc-mgs-pipeline/blob/main/GMH_pipe.py

Step 2: Profile the clades present in the samples (profileClades.sh)

#!/bin/bash

#SBATCH --mem=24gb
#SBATCH --time=0-07:59:59
#SBATCH --cpus-per-task=4
#SBATCH --open-mode=truncate
#SBATCH --job-name=SP4pr
#SBATCH --error=__SP4_profile.err
#SBATCH --output=__SP4_profile.out

# NOTES:
# script profiles all clades in the dataset in given folder ($1)
# puts results in the current folder!
# Adding --mutation_rates will give a mutation rates table for each of the alignes markers and a summary table for the concatenated MSA
# Removing the --print_clades only will actually run it 

# PARAMS
N=1 # --marker_in_n_samples
S=10 # --sample_with_n_markers
DB=/data/umcg-tifn/rgacesa/conda_biobakery4/lib/python3.10/site-packages/metaphlan/metaphlan_databases/mpa_vJan21_CHOCOPhlAnSGB_202103/mpa_vJan21_CHOCOPhlAnSGB_202103.pkl 

# purge modules
module purge
# load conda
ml Miniconda3/4.8.3
# load conda env
source activate /data/umcg-tifn/rgacesa/conda_biobakery4
# run clade profiling
strainphlan -s *.pkl --database /data/umcg-tifn/rgacesa/conda_biobakery4/lib/python3.10/site-packages/metaphlan/metaphlan_databases/mpa_vJan21_CHOCOPhlAnSGB_202103/mpa_vJan21_CHOCOPhlAnSGB_202103.pkl --marker_in_n_samples ${N} --sample_with_n_markers ${S} --print_clades_only --phylophlan_mode accurate --output_dir . > strainphlan4_clades_${N}.txt

Execution

sbatch ./profileClades.sh

Mon Mar 20 17:47:58 2023: Start StrainPhlAn 4.0.6 execution Mon Mar 20 17:47:58 2023: Loading MetaPhlAn mpa_vJan21_CHOCOPhlAnSGB_202103 database... Mon Mar 20 17:48:20 2023: Done. Mon Mar 20 17:48:23 2023: Detecting clades... Mon Mar 20 18:40:14 2023: Done. Mon Mar 20 18:40:14 2023: Detected clades: Mon Mar 20 18:40:14 2023: t__SGB10068: in 103 samples. Mon Mar 20 18:40:14 2023: t__SGB8007_group: in 86 samples. Mon Mar 20 18:40:14 2023: t__SGB6936: in 82 samples. Mon Mar 20 18:40:14 2023: t__SGB17248: in 77 samples. Mon Mar 20 18:40:14 2023: t__SGB6952: in 74 samples. Mon Mar 20 18:40:14 2023: t__SGB6939: in 73 samples. Mon Mar 20 18:40:14 2023: t__SGB9712_group: in 60 samples. Mon Mar 20 18:40:14 2023: t__SGB10120: in 58 samples. Mon Mar 20 18:40:14 2023: t__SGB10115: in 58 samples. Mon Mar 20 18:40:14 2023: t__SGB17247: in 56 samples. Mon Mar 20 18:40:14 2023: t__SGB7962: in 55 samples.

We next process this output file to select only the clade names

cat strainphlan4_clades_1.txt | grep t__ | cut -f 2 | cut -f 1 -d ':' > LLNEXT_sp_clades_names.txt

This will give us the names of each species found: t__SGB10068 t__SGB8007_group t__SGB6936 t__SGB17248 t__SGB6952 t__SGB6939 t__SGB9712_group t__SGB10120 t__SGB10115 t__SGB17247 t__S6_group t__SGB2303

Step 3: Build the multiple sequence alignment (sp4_runMarkerComparison.sh)

#!/bin/bash
#SBATCH --mem=24gb
#SBATCH --time=0-09:59:00
#SBATCH --cpus-per-task=8
#SBATCH --open-mode=truncate

# NOTES:
# > $1 is input folder
# > $2 is clade name

echo "Invoking runMarkerComparison.sh"
echo "CLs: ${1} ${2}"

# HELP / USE
if [[ $# -ne 2 ]]; 
    then 
    echo "ERROR: script requires two command line parameters:"
    echo " <input folder with pkl files> <clade name>"
    exit 2
fi

# PARAMS
# ===================
N=4 # --marker_in_n_samples
S=10 # --sample_with_n_markers 
MODE=accurate # {accurate,fast}
CONDA=/data/umcg-tifn/rgacesa/conda_biobakery4
#CM= # clade markers

# purge modules
module purge

# load conda
ml Miniconda3/4.8.3
# load conda env
source deactivate
source activate ${CONDA}

# prep results folder (where clade result goes)
mkdir ${2}
# run strainphlan for that clade
echo "strainphlan -s ${1}/*.pkl --output_dir ./${2} --clade ${2} --marker_in_n_samples ${N} --sample_with_n_markers ${S} --nprocs 8 --phylophlan_mode ${MODE}"
strainphlan -s ${1}/*.pkl --database /data/umcg-tifn/rgacesa/conda_biobakery4/lib/python3.10/site-packages/metaphlan/metaphlan_databases/mpa_vJan21_CHOCOPhlAnSGB_202103/mpa_vJan21_CHOCOPhlAnSGB_202103.pkl --output_dir ./${2} --clade ${2} --marker_in_n_samples ${N} --sample_with_n_markers ${S} --nprocs 8 --phylophlan_mode ${MODE} #--tmp ${OUT_TMP}

Execution

for i in $(cat CS_BABY_BIOME_clades_names.txt); do sbatch sp4_runMarkerComparison.sh  /scratch/umcg-tsinha/strainphlan_4_CS_BABY_BIOME $i; done

Where CS_BABY_BIOME_clades_names.txt contains a list of names of all (sub) species identified in the previous step This will perform MSA and create .tre files and .aln files for each of the (sub)species you feed it in

Step 4: Make distance matrix from MSA file (makeDistMatAll.sh)

#!/bin/bash
echo "maker of distance matrices"
echo " > goes through each folder and attempts to make distmat"

echo " > loading EMBOSS"
ml Anaconda3
source activate /home2/hb-llnext/EMBOSS_conda/ 
for F in */
do
   SN=${F/\/}
   echo ${SN}
   FM=${SN}.StrainPhlAn4_concatenated.aln
   echo "  >> making distmat"
   echo "distmat -sequence ${SN}/${FM} -nucmethod 2 -outfile ${SN}/${SN}.dmat"
distmat -sequence ${SN}/${FM} -nucmethod 2 -outfile ${SN}/${SN}.dmat
done   
 
# Creates a distance matrix from a multiple sequence alignment using the EMBOSS package (https://www.bioinformatics.nl/cgi-bin/emboss/help/distmat) 
# distmat calculates the evolutionary distance between every pair of sequences in a multiple sequence alignment.
# Uses Kimura Two-Parameter distance (distances expressed in terms of the number of substitutions per 100 b.p or amino acids)

Execution

sbatch makeDistMatAll.sh

Step 5: Cleaning the distance matrix from MSA file

First, load RPlus

ml RPlus

#Example: Rscript parseDMat_LLNext.R s__Bifidobacterium_bifidum.dmat

for i in $(find . -type f -name *.dmat); do Rscript parseDMAT.R $i; done

#parseDMAT.R consists of:

library(optparse)
# CL PARSER
help_description <- ""
args <- OptionParser(usage = "%prog clade.distmat.txt metadata.txt ordination.png",
                      add_help_option = TRUE, prog='strainphlan_ordination.R',
                      description=help_description )
args_list <- parse_args(args, positional_arguments=TRUE)

# read in the file, skipping the first 8 rows and filling in empty columns, using the tab as sep, and stripping extra white space
inFile <- args_list$args[1]
data <- read.table( inFile, skip = 8, fill = TRUE, sep="\t", strip.white = T)

# remove the first column of the data as it is blank
data[1] <- NULL

# get the header as the last column of the data as a character vector
header <- lapply(data[,ncol(data)], as.character)

# remove the last column from the data as it has been stored as a header
data[ncol(data)] <- NULL

# remove the current last column from the data as it is blank
data[ncol(data)] <- NULL

# split header by space and digit to get the sample names
samples <- unlist(header)
# fix metaphlan just by taking only first 3 thingies after _ split
ss <- c()
sNR <- 0
for (s in samples) {
   sNR <- sNR + 1
   ssplit = strsplit(s,' ')
   ss <- c(ss,ssplit[[1]][1])
}

# add the sample names to the columns and rows of the data matrix
rownames(data) <- ss
colnames(data) <- ss

# make symmetric, add lower triangle to upper triangle
data[lower.tri(data)] <- t(data)[lower.tri(data)]

# save it
write.table(data,paste0(gsub('\\.dmat','',inFile),'_dmat_Rready.csv'),sep=',',row.names=T)