diff --git a/.gitignore b/.gitignore index 626eb6b7ed3047d056a519fc0a6dcfd27c6feea8..2defeaab3edf4195e3c17e6fd3fc76fb9c9a5536 100644 --- a/.gitignore +++ b/.gitignore @@ -1 +1,4 @@ -pangenome \ No newline at end of file +pangenome +assembly/container/assemblySC.sif +assembly/.snakemake +assembly/rulegraph.pdf \ No newline at end of file diff --git a/README.md b/README.md index 99fb3fc9112b551593d4d7363fd80fe76d9948ca..a1293f5307867828143b7bc0108a3607fdbd6cb8 100644 --- a/README.md +++ b/README.md @@ -1,8 +1,8 @@ # Genome assembly and variant calling from PacBio HiFi reads This folder contains two Snakemake workflows: - - *assembly*: from PacBio HiFi consensus reads to annotated genome assemblies - - *variantcalling*: combine assemblies into a pangenome graph and call variants from the graph + - [*assembly*](assembly/README.md): from PacBio HiFi consensus reads to annotated genome assemblies + - [*variantcalling*](variantcalling/README.md): combine assemblies into a pangenome graph and call variants from the graph This is ongoing work, some things will change. @@ -14,7 +14,7 @@ ml snakemake/6.6.1-foss-2021a ml Singularity/2.6.1 ``` -Enter subdirectories for further information. +<img src="fig.png" width="50%" height="50%"> + -#<img src="fig.png" width="50%" height="50%"> *Section of a genome graph constructed from diverse MTBC genomes. It shows a nested structural variant: an IS6110 insertion within the TBD1 region.* diff --git a/assembly/config.yaml b/assembly/config/config.yaml old mode 100755 new mode 100644 similarity index 89% rename from assembly/config.yaml rename to assembly/config/config.yaml index ae05f92d44be64bbaa79f1ca0b62ce9ff9c77375..6b82edb7a11e09446e7191e42a029efdabee70dc --- a/assembly/config.yaml +++ b/assembly/config/config.yaml @@ -2,10 +2,9 @@ # ############################## -samples: samples.tsv - project_directory: ./results output_prefix: pb_bernese +samples: config/samples.tsv ref: genome_size: 4.4m diff --git a/assembly/samples.tsv b/assembly/config/samples.tsv old mode 100755 new mode 100644 similarity index 100% rename from assembly/samples.tsv rename to assembly/config/samples.tsv diff --git a/assembly/container/README.md b/assembly/container/README.md index 9d4c1e4a3c2da5f15c022bf2b0ad04ceb36dc150..83dbaae8f9adf1e2ad0ae042dff50d18f6b29cc6 100644 --- a/assembly/container/README.md +++ b/assembly/container/README.md @@ -1,17 +1,4 @@ -Problem: 1.6 gb container on gitlab? Building or loading container requires sudo... - - -``` -sudo singularity build assemblySC.sif assemblySC.def -``` - - - - - - - # Create versioned direct file and locked dependency file (https://pythonspeed.com/articles/conda-dependency-management/) environment.yml: versioned main dependencies conda-lock.yml: locked subdependencies @@ -24,7 +11,6 @@ mamba install \ samtools \ minimap2 \ circlator \ - panaroo \ prokka \ numpy \ scipy \ @@ -47,3 +33,9 @@ conda env update --file environment.yml --prune # Create locked version conda-lock --mamba -f environment.yml -p linux-64 conda-lock render -p linux-64 # allows using mamba create --file conda-linux-64.lock + +Problem: 1.6 gb container on gitlab? Building or loading container requires sudo... + +``` +sudo singularity build assemblySC.sif assemblySC.def +``` \ No newline at end of file diff --git a/assembly/container/assemblySC.def b/assembly/container/assemblySC.def index be3b4c236c207ba1d794520fc8bb2dccc4c279ae..51e86dedb021a39b59dabb8f53539e5e1c82df9e 100644 --- a/assembly/container/assemblySC.def +++ b/assembly/container/assemblySC.def @@ -8,7 +8,7 @@ From: condaforge/mambaforge:23.1.0-1 export PATH=/opt/conda/bin:$PATH - mamba create --name pb_assembly --file conda-linux-64.lock + mamba create --name assemblySC --file conda-linux-64.lock mamba clean -afy apt-get clean all @@ -22,21 +22,21 @@ From: condaforge/mambaforge:23.1.0-1 cd LongQC/minimap2-coverage && make # Install mash - apt-get install -y mash + #apt-get install -y mash # Install polypolish - wget https://github.com/rrwick/Polypolish/releases/download/v0.5.0/polypolish-linux-x86_64-musl-v0.5.0.tar.gz - tar -xf polypolish-linux-x86_64-musl-v0.5.0.tar.gz -C /opt/conda/bin - rm polypolish-linux-x86_64-musl-v0.5.0.tar.gz + #wget https://github.com/rrwick/Polypolish/releases/download/v0.5.0/polypolish-linux-x86_64-musl-v0.5.0.tar.gz + #tar -xf polypolish-linux-x86_64-musl-v0.5.0.tar.gz -C /opt/conda/bin + #rm polypolish-linux-x86_64-musl-v0.5.0.tar.gz # Install progressiveCactus - wget https://github.com/ComparativeGenomicsToolkit/cactus/releases/download/v2.6.0/cactus-bin-v2.6.0.tar.gz - tar -xf cactus-bin-v2.6.0.tar.gz -C /opt/conda/bin - rm cactus-bin-v2.6.0.tar.gz + #wget https://github.com/ComparativeGenomicsToolkit/cactus/releases/download/v2.6.0/cactus-bin-v2.6.0.tar.gz + #tar -xf cactus-bin-v2.6.0.tar.gz -C /opt/conda/bin + #rm cactus-bin-v2.6.0.tar.gz # set to whatever your conda path is, I usually install to /opt echo ". /opt/conda/etc/profile.d/conda.sh" >> $SINGULARITY_ENVIRONMENT - echo "conda activate pb_assembly" >> $SINGULARITY_ENVIRONMENT + echo "conda activate assemblySC" >> $SINGULARITY_ENVIRONMENT %environment diff --git a/assembly/container/conda-linux-64.lock b/assembly/container/conda-linux-64.lock index c09a9a3a5c489ac52771ef55751c0d15e26a2666..7a872e3ed42f97c65dec979c6a9f988eafc100f5 100644 --- a/assembly/container/conda-linux-64.lock +++ b/assembly/container/conda-linux-64.lock @@ -1,372 +1,279 @@ # Generated by conda-lock. # platform: linux-64 -# input_hash: 182af84a8c8a1dc9eb2c6bdc76285050405b5c964d7feda7b0a4ea7d966f0976 +# input_hash: 926ab17aedcc8b15a4be8db176a2c48cafc2fcb2c9f778737a63eb301e4e346e @EXPLICIT -https://repo.anaconda.com/pkgs/main/linux-64/_libgcc_mutex-0.1-main.conda#c3473ff8bdb3d124ed5ff11ec380d6f9 -https://repo.anaconda.com/pkgs/main/linux-64/blas-1.0-mkl.conda#9a7a051e9bd41da46523acb017d8a517 -https://repo.anaconda.com/pkgs/main/linux-64/ca-certificates-2023.05.30-h06a4308_0.conda#979be8dd2368decd342b13e01540d297 +https://conda.anaconda.org/conda-forge/linux-64/_libgcc_mutex-0.1-conda_forge.tar.bz2#d7c89558ba9fa0495403155b64376d81 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perl-bioperl: '>=1.7.2' + perl-xml-simple: '' + prodigal: '>=2.6' + tbl2asn-forever: '>=25.7' + hash: + md5: 6aea788d2ca74ab50d6816e7e0a75da2 + sha256: 654e7ab399907d0153052dc2f729d4b8db5de6c819686d2dbb8a4890782595c4 + manager: conda + name: prokka + optional: false + platform: linux-64 + url: https://conda.anaconda.org/bioconda/noarch/prokka-1.14.6-pl5321hdfd78af_5.tar.bz2 + version: 1.14.6 +version: 1 diff --git a/assembly/container/environment.yml b/assembly/container/environment.yml index b9973f8b4e9ab3e52ee37a0a715a4d7ed4a2d2c4..cbebc99fe5b6188e4ad1fa532b91819b10d15387 100644 --- a/assembly/container/environment.yml +++ b/assembly/container/environment.yml @@ -1,8 +1,9 @@ -name: pb_assembly +name: assemblySC channels: + - defaults + - conda-forge - bioconda - r - - defaults dependencies: - flye - samtools @@ -20,4 +21,7 @@ dependencies: - edlib - python-edlib - biopython -prefix: /home/cristobal/anaconda3/envs/pb_assembly + - ca-certificates + - certifi + - openssl +prefix: /home/cristobal/anaconda3/envs/assemblySC diff --git a/assembly/resources/H37Rv.gbf b/assembly/resources/H37Rv.gbf old mode 100755 new mode 100644 diff --git a/assembly/workflow/rules/annotate.smk b/assembly/workflow/rules/annotate.smk old mode 100755 new mode 100644 diff --git a/assembly/workflow/rules/assemble.smk b/assembly/workflow/rules/assemble.smk old mode 100755 new mode 100644 diff --git a/assembly/workflow/rules/circularize.smk b/assembly/workflow/rules/circularize.smk old mode 100755 new mode 100644 index 17a10f1e83e31d8a83b2da85e7efff29c45939d7..cce5cb3c333e117c913a5b61431727c5f7f1cea7 --- a/assembly/workflow/rules/circularize.smk +++ b/assembly/workflow/rules/circularize.smk @@ -1,5 +1,5 @@ -rule mapreads: +rule circlator_mapreads: input: assembly = 'results/{sample}/assembly.fasta', reads = lambda wildcards: expand(samples[wildcards.sample].longread_fastq) @@ -13,7 +13,7 @@ rule mapreads: """ -rule bam2reads: +rule circlator_bam2reads: input: "results/{sample}/circlator/01.mapreads.bam" output: "results/{sample}/circlator/02.bam2reads.fasta" params: @@ -24,7 +24,7 @@ rule bam2reads: """ -rule localassembly: +rule circlator_localassembly: input: "results/{sample}/circlator/02.bam2reads.fasta" output: "results/{sample}/circlator/03.assemble/assembly.fasta" params: @@ -36,7 +36,7 @@ rule localassembly: """ -rule merge: +rule circlator_merge: input: assembly = 'results/{sample}/assembly.fasta', localassembly = "results/{sample}/circlator/03.assemble/assembly.fasta" @@ -50,7 +50,7 @@ rule merge: """ -rule clean: +rule circlator_clean: input: "results/{sample}/circlator/04.merge.fasta" output: "results/{sample}/circlator/05.clean.fasta" params: @@ -61,7 +61,7 @@ rule clean: """ -rule fixstart: +rule circlator_fixstart: input: "results/{sample}/circlator/05.clean.fasta" output: "results/{sample}/circlator/06.fixstart.fasta" params: diff --git a/assembly/workflow/rules/common.smk b/assembly/workflow/rules/common.smk old mode 100755 new mode 100644 index 63ccf25c8dfd65753d8a4c11ce0599dab46f8d80..eac2fbc0f20255a5105cdacfd40dd26496a3afe4 --- a/assembly/workflow/rules/common.smk +++ b/assembly/workflow/rules/common.smk @@ -1,4 +1,4 @@ -configfile: "config.yaml" +configfile: "config/config.yaml" class sample: def __init__(self, fields): diff --git a/assembly/workflow/rules/readQC.smk b/assembly/workflow/rules/readQC.smk old mode 100755 new mode 100644 diff --git a/variantcalling/README.md b/variantcalling/README.md index a3424f8d6b1849c468068feb1a5f254c39bb5316..ce3013ac0eb8affd8714990f28e98e3a9a2f3344 100644 --- a/variantcalling/README.md +++ b/variantcalling/README.md @@ -3,23 +3,18 @@ ## assemblies.txt - - -## config.yaml +## config/config.yaml ```yaml -samples: samples.inhouse.hifi.test.csv - -results_directory: ./results -output_prefix: pb_bernese - -ref: - genome_size: 4.4m - gbf: resources/H37Rv.gbf +reference: threads: 4 -keep_intermediate: "Yes" +output_dir: ./results + +pggb: + p: 99 + s: 5k ``` diff --git a/variantcalling/config/biopython.yaml b/variantcalling/config/biopython.yaml new file mode 100644 index 0000000000000000000000000000000000000000..9b7042f57eb6b06206ba966c3ab73e3d590400aa --- /dev/null +++ b/variantcalling/config/biopython.yaml @@ -0,0 +1,40 @@ +name: biopython +channels: + - defaults + - conda-forge + - bioconda + - r +dependencies: + - _libgcc_mutex=0.1=main + - _openmp_mutex=5.1=1_gnu + - biopython=1.78=py311h5eee18b_0 + - blas=1.0=mkl + - bzip2=1.0.8=h7b6447c_0 + - ca-certificates=2023.08.22=h06a4308_0 + - intel-openmp=2023.1.0=hdb19cb5_46305 + - ld_impl_linux-64=2.38=h1181459_1 + - libffi=3.4.4=h6a678d5_0 + - libgcc-ng=11.2.0=h1234567_1 + - libgomp=11.2.0=h1234567_1 + - libstdcxx-ng=11.2.0=h1234567_1 + - libuuid=1.41.5=h5eee18b_0 + - mkl=2023.1.0=h213fc3f_46343 + - mkl-service=2.4.0=py311h5eee18b_1 + - mkl_fft=1.3.8=py311h5eee18b_0 + - mkl_random=1.2.4=py311hdb19cb5_0 + - ncurses=6.4=h6a678d5_0 + - numpy=1.26.0=py311h08b1b3b_0 + - numpy-base=1.26.0=py311hf175353_0 + - openssl=3.0.11=h7f8727e_2 + - pip=23.2.1=py311h06a4308_0 + - python=3.11.5=h955ad1f_0 + - readline=8.2=h5eee18b_0 + - setuptools=68.0.0=py311h06a4308_0 + - sqlite=3.41.2=h5eee18b_0 + - tbb=2021.8.0=hdb19cb5_0 + - tk=8.6.12=h1ccaba5_0 + - tzdata=2023c=h04d1e81_0 + - wheel=0.41.2=py311h06a4308_0 + - xz=5.4.2=h5eee18b_0 + - zlib=1.2.13=h5eee18b_0 +prefix: /home/cristobal/anaconda3/envs/biopython diff --git a/variantcalling/config.yaml b/variantcalling/config/config.yaml similarity index 100% rename from variantcalling/config.yaml rename to variantcalling/config/config.yaml diff --git a/variantcalling/samples.txt b/variantcalling/config/samples.txt similarity index 100% rename from variantcalling/samples.txt rename to variantcalling/config/samples.txt diff --git a/variantcalling/workflow/Snakefile b/variantcalling/workflow/Snakefile index ff09382699d3de1a26f0a2651659a12ea91d304c..cc39245ce400f64986af595a1ab0d4464165df94 100644 --- a/variantcalling/workflow/Snakefile +++ b/variantcalling/workflow/Snakefile @@ -31,32 +31,13 @@ rule combine_assemblies: output: fasta = "results/single_contig_assemblies.fasta", discarded = "results/discarded_assemblies.txt" - conda: "env.yaml" + conda: "config/biopython.yaml" shell: """ - python ./combine_assemblies.py {input} + python workflow/scripts/combine_assemblies.py {input} """ - run: - import sys - from Bio import SeqIO - - fasta_handle = open(output.fasta, "w") - discarded = open(output.discarded, "w") - - for ASSEMBLY in input: - - records = SeqIO.parse(ASSEMBLY, "fasta") - if len(records) > 1: - discarded.write(ASSEMBLY + '\n') - - else: - rec = next(records) - SeqIO.write(rec, fasta_handle, "fasta") - - fasta_handle.close() - discarded.close() - + rule pggb: input: "results/single_contig_assemblies.fasta" diff --git a/variantcalling/workflow/scripts/combine_assemblies.py b/variantcalling/workflow/scripts/combine_assemblies.py new file mode 100644 index 0000000000000000000000000000000000000000..39321570a9e9d355b9e249789fc125b40a2c4a34 --- /dev/null +++ b/variantcalling/workflow/scripts/combine_assemblies.py @@ -0,0 +1,25 @@ +#!/usr/bin/env python3 +# -*- coding: utf-8 -*- + +import re +import sys +from Bio import SeqIO + + +assemblies = sys.argv[1:] + +fasta_handle = open("results_sim/single_contig_assemblies.fasta", "w") +discarded = open("results_sim/discarded_assemblies.txt", "w") + +for ASSEMBLY in assemblies: + + records = [rec for rec in SeqIO.parse(ASSEMBLY, "fasta")] + if len(records) > 1: + discarded.write(ASSEMBLY + '\n') + + else: + rec = records[0] + SeqIO.write(rec, fasta_handle, "fasta") + +fasta_handle.close() +discarded.close() \ No newline at end of file