diff --git a/notebooks/create_pangenome_graph.ipynb b/notebooks/create_pangenome_graph.ipynb
new file mode 100644
index 0000000000000000000000000000000000000000..8ec974a6a0b26207400cec6dec0ac44537da2cae
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+++ b/notebooks/create_pangenome_graph.ipynb
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+{
+ "cells": [
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "# Create a pangenome graph with pggb\n",
+ "\n",
+ "pggb requires a single gzipped and indexed fasta file containing all assemblies. "
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "## Install biopython\n",
+ "\n",
+ "If not already present, create a conda environment with biopython. \n",
+ "\n",
+ "```\n",
+ "conda create -n bio biopython\n",
+ "```"
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "Then select the bio environent as the kernel for the Jupyter notebook."
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "## Combine assemblies\n",
+ "\n",
+ "The script below creates a file 'assemblies_combined.fasta' in which all assemblies listed in 'paths_to_assemblies.txt' are combined.\n",
+ "\n",
+ "'paths_to_assemblies.txt' should be a text file with one path per line. \n",
+ "\n",
+ "If one_contig_only is set to True, only single-contig assemblies are included."
+ ]
+ },
+ {
+ "cell_type": "code",
+ "execution_count": null,
+ "metadata": {},
+ "outputs": [],
+ "source": [
+ "from Bio import SeqIO\n",
+ "\n",
+ "\n",
+ "assemblies = 'paths_to_assemblies.txt'\n",
+ "one_contig_only = False\n",
+ "\n",
+ "fasta_combined = []\n",
+ "\n",
+ "with open(assemblies) as f:\n",
+ " \n",
+ " for line in f:\n",
+ " \n",
+ " assembly_path = line.strip()\n",
+ " strain = assembly_path.split('/')[-1].split('.')[0]\n",
+ " \n",
+ " records = [rec for rec in SeqIO.parse(assembly_path, \"fasta\")]\n",
+ " \n",
+ " if one_contig_only and len(records) > 1:\n",
+ " print('Discarded multi-contig assembly for ' + strain)\n",
+ " continue\n",
+ " \n",
+ " fasta_combined += records\n",
+ "\n",
+ "SeqIO.write(fasta_combined, 'assemblies_combined.fasta', 'fasta')\n",
+ "\n"
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "## Zip and index fasta"
+ ]
+ },
+ {
+ "cell_type": "code",
+ "execution_count": null,
+ "metadata": {
+ "vscode": {
+ "languageId": "shellscript"
+ }
+ },
+ "outputs": [],
+ "source": [
+ "%%bash\n",
+ "\n",
+ "gzip single_contig_assemblies.fasta\n",
+ "samtools faidx single_contig_assemblies.fasta.gz"
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "## Run pggb\n",
+ "\n",
+ "Important paramters:\n",
+ "\n",
+ " -i : path to combined assemblies\n",
+ " -o : output directory name\n",
+ " -n : Number of contigs included\n",
+ " -p : Similarity of contigs\n",
+ " -s : Maximum length of repeats in the genome"
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "Example slurm script:"
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "#!/bin/bash\n",
+ "\n",
+ "#SBATCH --cpus-per-task=20\n",
+ "#SBATCH --mem-per-cpu=4G\n",
+ "#SBATCH --time=24:00:00\n",
+ "#SBATCH --qos=1day\n",
+ "#SBATCH --output=stdout.%j\n",
+ "#SBATCH --error=stderr.%j\n",
+ "\n",
+ "singularity exec /scicore/home/gagneux/GROUP/PacbioSnake_resources/containers/pggb_latest.sif pggb \\\n",
+ " -i assemblies_combined.fasta.gz \\\n",
+ " -o ./pggb \\\n",
+ " -m \\\n",
+ " -t 20 \\\n",
+ " -n 52 \\\n",
+ " -p 99 \\\n",
+ " -s 5k\n"
+ ]
+ }
+ ],
+ "metadata": {
+ "kernelspec": {
+ "display_name": "Python 3",
+ "language": "python",
+ "name": "python3"
+ },
+ "language_info": {
+ "codemirror_mode": {
+ "name": "ipython",
+ "version": 3
+ },
+ "file_extension": ".py",
+ "mimetype": "text/x-python",
+ "name": "python",
+ "nbconvert_exporter": "python",
+ "pygments_lexer": "ipython3",
+ "version": "3.10.12"
+ }
+ },
+ "nbformat": 4,
+ "nbformat_minor": 2
+}
diff --git a/notebooks/pangenome_graph.ipynb b/notebooks/pangenome_graph.ipynb
deleted file mode 100644
index e69de29bb2d1d6434b8b29ae775ad8c2e48c5391..0000000000000000000000000000000000000000
diff --git a/notebooks/tree_from_graph.ipynb b/notebooks/tree_from_graph.ipynb
new file mode 100644
index 0000000000000000000000000000000000000000..b3052943781b741185276bd090f1b7a182ef2874
--- /dev/null
+++ b/notebooks/tree_from_graph.ipynb
@@ -0,0 +1,310 @@
+{
+ "cells": [
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "# Create a phylogenetic tree from a pangenome graph\n",
+ "\n",
+ "Call SNPs from graph with vg deconstruct.\n",
+ "\n",
+ "Filter out resistance genes and clustered variants indicative of gene conversion or selection acting on sloppy targets.\n",
+ "\n",
+ "Maybe determine clustering threshold through simulations?"
+ ]
+ },
+ {
+ "cell_type": "code",
+ "execution_count": null,
+ "metadata": {
+ "vscode": {
+ "languageId": "plaintext"
+ }
+ },
+ "outputs": [],
+ "source": [
+ "%%bash\n",
+ "\n",
+ "singularity exec /home/cristobal/programs/pggb_latest.sif vg deconstruct \\\n",
+ " pggb/assemblies_combined.fasta.gz.d71a954.11fba48.a4da63a.smooth.final.gfa -d1 -e \\\n",
+ " -p H37Rv \\\n",
+ " -t 4 \\\n",
+ " --all-snarls \\\n",
+ " > variants.vcf\n",
+ "\n",
+ "grep -v -c ^# variants.vcf"
+ ]
+ },
+ {
+ "cell_type": "code",
+ "execution_count": null,
+ "metadata": {},
+ "outputs": [],
+ "source": [
+ "# Create list of positions to exclude\n",
+ "import pandas\n",
+ "\n",
+ "dr_loci = pandas.read_csv(\"DR_genes_coordinates.tsv\", sep=\"\\t\")\n",
+ "repeats = pandas.read_csv(\"nucmer/delta.out.filtered.tsv\")\n",
+ "\n",
+ "excluded = set()\n",
+ "\n",
+ "for i, gene in dr_loci.iterrows():\n",
+ " excluded.update(range(gene[\"start_region\"], gene[\"stop_region\"]+1))\n",
+ "\n",
+ "for i,hpair in repeats.iterrows():\n",
+ " excluded.update(range(hpair[\"S1\"], hpair[\"E1\"]+1))\n",
+ " excluded.update(range(hpair[\"S2\"], hpair[\"E2\"]+1))"
+ ]
+ },
+ {
+ "cell_type": "code",
+ "execution_count": null,
+ "metadata": {},
+ "outputs": [],
+ "source": [
+ "canettii = [\"CIPT_140010059_/_STB-A\", \"ET1291\"]\n",
+ "\n",
+ "keep = []\n",
+ "\n",
+ "discarded = {\n",
+ " \"In repeat or DR locus\" : 0,\n",
+ " \"Not SNP\" : 0,\n",
+ " \"canettii only\" : 0,\n",
+ " \"Missing genotypes\" : 0,\n",
+ " \"Invariant\" : 0\n",
+ "}\n",
+ "\n",
+ "with open(\"variants.vcf\") as f:\n",
+ " \n",
+ " for line in f:\n",
+ " \n",
+ " if line.startswith(\"#CHROM\"):\n",
+ " \n",
+ " fields = line.strip().split(\"\\t\")\n",
+ " strains = fields[9:]\n",
+ " canettii_i = [strains.index(x) for x in canettii]\n",
+ " \n",
+ " # Dictionary to store SNPs for each strain\n",
+ " strain_seqs = {strain : \"\" for strain in strains}\n",
+ " strain_seqs[\"H37Rv\"] = \"\"\n",
+ " continue\n",
+ " \n",
+ " elif line.startswith(\"#\"):\n",
+ " continue\n",
+ "\n",
+ " fields = line.strip().split(\"\\t\")\n",
+ " position = int(fields[1])\n",
+ " \n",
+ " ref = fields[3]\n",
+ " alt = fields[4].split(\",\")\n",
+ " \n",
+ " gt = fields[9:]\n",
+ " \n",
+ " # Two entries weirdly have missing genotypes. Skip them\n",
+ " if len(gt) != len(strains):\n",
+ " print(fields)\n",
+ " continue\n",
+ " \n",
+ " \n",
+ " canettii_gt = [gt[i] for i in canettii_i]\n",
+ " others = [gt[i] for i in range(len(gt)) if i not in canettii_i]\n",
+ " \n",
+ " \n",
+ " # Exclude sites in repeats and DR loci\n",
+ " if position in excluded:\n",
+ " discarded[\"In repeat or DR locus\"] += 1\n",
+ " continue\n",
+ "\n",
+ " # Only SNPs\n",
+ " if len(ref) > 1 or any(len(x) > 1 for x in alt):\n",
+ " discarded[\"Not SNP\"] += 1\n",
+ " continue\n",
+ " \n",
+ " # Skip canettii-only variants\n",
+ " if \"1\" in canettii_gt and \"1\" not in others:\n",
+ " discarded[\"canettii only\"] += 1\n",
+ " continue\n",
+ "\n",
+ " # Exclude missing genotypes\n",
+ " if any(x in gt for x in [\"\", \".\"]):\n",
+ " discarded[\"Missing genotypes\"] += 1\n",
+ " #continue\n",
+ "\n",
+ "\n",
+ " # Write alleles to site\n",
+ " alleles = [ref] + alt \n",
+ " site = ''\n",
+ " \n",
+ " for strain in strains:\n",
+ " strain_i = strains.index(strain)\n",
+ " strain_gt = gt[strain_i]\n",
+ " if not strain_gt or strain_gt not in '012345':\n",
+ " strain_allele = '-'\n",
+ " else:\n",
+ " strain_allele = alleles[int(strain_gt)]\n",
+ " \n",
+ " site += strain_allele\n",
+ " \n",
+ " # Re-check if there are remaining invariant sites\n",
+ " bases_only = ''\n",
+ " for allele in site:\n",
+ " if allele in ['A','C','G', 'T']:\n",
+ " bases_only += allele\n",
+ "\n",
+ " #bases_only = ref + site.replace('-', '')\n",
+ " \n",
+ " \n",
+ " if len(set(bases_only)) > 1:\n",
+ " \n",
+ " for i, allele in enumerate(site):\n",
+ " strain = strains[i]\n",
+ " strain_seqs[strain] += allele\n",
+ " \n",
+ " # Write H37Rv allele\n",
+ " strain_seqs[\"H37Rv\"] += ref\n",
+ " \n",
+ " keep.append((position, alleles, gt))\n",
+ " \n",
+ " else:\n",
+ " discarded[\"Invariant\"] += 1\n",
+ "\n",
+ " \n",
+ " \n",
+ "print(\"Filtered out:\")\n",
+ "for k in discarded:\n",
+ " print(k, str(discarded[k]))\n",
+ " \n",
+ "print(\"Kept: \", str(len(keep)))"
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "Write variable alignment to fasta"
+ ]
+ },
+ {
+ "cell_type": "code",
+ "execution_count": null,
+ "metadata": {},
+ "outputs": [],
+ "source": [
+ "from Bio import SeqIO\n",
+ "from Bio.Seq import Seq\n",
+ "from Bio.SeqRecord import SeqRecord\n",
+ "\n",
+ "records = []\n",
+ "\n",
+ "for strain in [\"H37Rv\"] + strains:\n",
+ " \n",
+ " sequence = Seq(strain_seqs[strain])\n",
+ " record = SeqRecord(sequence, id=strain, name=\"\", description=\"\")\n",
+ " records.append(record)\n",
+ " \n",
+ "SeqIO.write(records, \"H37Rv_SNP_alignment.from_graph.fasta\", \"fasta\")\n"
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "## Count non-variable positions\n",
+ "\n",
+ "Get the core nodes from the graph and count the number of A,C,G,T"
+ ]
+ },
+ {
+ "cell_type": "code",
+ "execution_count": null,
+ "metadata": {},
+ "outputs": [],
+ "source": [
+ "from collections import Counter\n",
+ "\n",
+ "GRAPH='/home/cristobal/TB/projects/deletionism/NOTEBOOKS/A_ancestral_reference/pggb/assemblies_combined.fasta.gz.d71a954.11fba48.a4da63a.smooth.final.gfa'\n",
+ "\n",
+ "n_paths = 0\n",
+ "\n",
+ "node_seqs = {}\n",
+ "node_count = {}\n",
+ "\n",
+ "# First traversal: get nodes present in all paths\n",
+ "with open(GRAPH) as f:\n",
+ " for line in f:\n",
+ " \n",
+ " fields = line.strip().split('\\t')\n",
+ " \n",
+ " if line.startswith('S'):\n",
+ " node_seqs[fields[1]] = fields[2]\n",
+ " \n",
+ " elif line.startswith('P'):\n",
+ " \n",
+ " n_paths += 1\n",
+ " \n",
+ " paff = [x[:-1] for x in fields[2].split(',') ]\n",
+ " \n",
+ " for node in paff: \n",
+ " \n",
+ " if node not in node_count:\n",
+ " node_count[node] = 0\n",
+ " \n",
+ " node_count[node] += 1\n",
+ " \n",
+ "invariable_seq = ''\n",
+ "\n",
+ "for node in node_count:\n",
+ " if node_count[node] == n_paths:\n",
+ " invariable_seq += node_seqs[node]\n",
+ " \n",
+ "base_counts = Counter(invariable_seq)\n",
+ "\n",
+ "for base in base_counts:\n",
+ " print(base, base_counts[base])\n",
+ "\n",
+ "\n",
+ "total = sum([base_counts[base] for base in base_counts])\n",
+ "gc = base_counts['G'] + base_counts['C']\n",
+ "\n",
+ "print('Total invariable bases: ', total)\n",
+ "print('GC content: ', gc/total)\n"
+ ]
+ },
+ {
+ "cell_type": "markdown",
+ "metadata": {},
+ "source": [
+ "## Estimate tree!\n",
+ "\n",
+ "Use Stamatakis model in RAXML to account for invariable sites and correct branch lengths.\n",
+ "\n",
+ "--model GTR+G+ASC_STAM{633511/1206483/1214591/640523}"
+ ]
+ },
+ {
+ "cell_type": "code",
+ "execution_count": null,
+ "metadata": {},
+ "outputs": [],
+ "source": [
+ "%%bash\n",
+ "\n",
+ "~/programs/raxml-ng_v1.2.0_linux_x86_64/raxml-ng \\\n",
+ " --msa H37Rv_SNP_alignment.from_graph.fasta \\\n",
+ " --all --model GTR+G+ASC_STAM{633312/1206359/1214398/640320} --tree pars{10} --bs-trees 100 --threads 4 --outgroup CIPT_140010059_/_STB-A\n",
+ "\n",
+ "mv H37Rv_SNP_alignment.from_graph.fasta.* raxml\n",
+ "\n",
+ " "
+ ]
+ }
+ ],
+ "metadata": {
+ "language_info": {
+ "name": "python"
+ }
+ },
+ "nbformat": 4,
+ "nbformat_minor": 2
+}