Commit ac18b0b9 authored by Weipeng Mo's avatar Weipeng Mo
Browse files

update snakefile for reproducibility.

parent 1aa89fac

README.md

+8 −1
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@@ -21,6 +21,13 @@ This pipeline includes basecalling and preprocessing of the FLEP-seq data. 
  * Tidyverse (https://www.tidyverse.org/)

  * optparse (https://cran.r-project.org/web/packages/optparse/index.html)



**One-step installation**



Install FLEP-seq pipeline, its dependencies, and other required packages in one step using conda or mamba:



```

mamba create -n flepseq minimap2 pysam click ont-fast5-api joblib r-tidyverse r-optparse samtools pandas

```



## Inputs

environment.yml

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name: flepseq

channels:

  - bioconda

  - conda-forge

  - defaults

dependencies:

  - _libgcc_mutex=0.1=conda_forge

  - _openmp_mutex=4.5=2_gnu

  - _r-mutex=1.0.1=anacondar_1

  - binutils_impl_linux-64=2.40=hf600244_0

  - bwidget=1.9.14=ha770c72_1

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  - ca-certificates=2023.01.10=h06a4308_0

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  - cached_property=1.5.2=pyha770c72_1

  - cairo=1.16.0=ha61ee94_1014

  - click=8.1.3=py310hff52083_1

  - curl=7.87.0=h6312ad2_0

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  - gxx_impl_linux-64=12.2.0=hcc96c02_19

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  - liblapack=3.9.0=16_linux64_openblas

  - libnghttp2=1.51.0=hdcd2b5c_0

  - libnsl=2.0.0=h7f98852_0

  - libopenblas=0.3.21=pthreads_h78a6416_3

  - libpng=1.6.39=h753d276_0

  - libsanitizer=12.2.0=h46fd767_19

  - libsqlite=3.40.0=h753d276_0

  - libssh2=1.10.0=haa6b8db_3

  - libstdcxx-devel_linux-64=12.2.0=h3b97bd3_19

  - libstdcxx-ng=12.2.0=h46fd767_19

  - libtiff=4.4.0=h0e0dad5_3

  - libuuid=2.38.1=h0b41bf4_0

  - libwebp-base=1.3.0=h0b41bf4_0

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  - make=4.3=hd18ef5c_1

  - minimap2=2.24=h7132678_1

  - ncurses=6.4=h6a678d5_0

  - numpy=1.24.2=py310h8deb116_0

  - ont-fast5-api=4.1.1=pyhdfd78af_0

  - openssl=1.1.1t=h0b41bf4_0

  - packaging=23.1=pyhd8ed1ab_0

  - pandas=2.0.0=py310h9b08913_0

  - pandoc=2.19.2=h32600fe_2

  - pango=1.50.14=hd33c08f_0

  - pcre2=10.40=hc3806b6_0

  - pip=23.0.1=pyhd8ed1ab_0

  - pixman=0.40.0=h36c2ea0_0

  - progressbar33=2.4=py_0

  - pthread-stubs=0.4=h36c2ea0_1001

  - pysam=0.21.0=py310hff46b53_0

  - python=3.10.8=h257c98d_0_cpython

  - python-dateutil=2.8.2=pyhd8ed1ab_0

  - python-tzdata=2023.3=pyhd8ed1ab_0

  - python_abi=3.10=3_cp310

  - pytz=2023.3=pyhd8ed1ab_0

  - r-askpass=1.1=r42h06615bd_3

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  - r-digest=0.6.31=r42h38f115c_0

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  - r-fansi=1.0.4=r42h133d619_0

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  - r-htmltools=0.5.5=r42h38f115c_0

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  - r-lubridate=1.9.2=r42h133d619_1

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  - r-openssl=2.0.5=r42hb1dc35e_0

  - r-optparse=1.7.3=r42hc72bb7e_1

  - r-pillar=1.9.0=r42hc72bb7e_0

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  - r-sys=3.4.1=r42h06615bd_0

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prefix: /work/bio-mowp/anaconda3/envs/flepseq

requirements.txt

+4 −1
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minimap2

pysam

numpy

pandas

samtools

click

ont-fast5-api

joblib

run_guppy.sh

0 → 100644
+19 −0
Original line number Diff line number Diff line 
#BSUB -J 01_guppy

#BSUB -n 20

#BSUB -o %J.stdout

#BSUB -e %J.stderr

#BSUB -R span[hosts=1]

#BSUB -q gpu





export PATH=/work/bio-mowp/software/ont-guppy-4.0.11/bin:$PATH



guppy_basecaller \

  -i fast5 \

  -s guppy_out \

  -c dna_r9.4.1_450bps_hac.cfg \

  --recursive \

  --disable_pings \

  --fast5_out \

  --qscore_filtering \

  --device "cuda:all:100%"

snakefile

+9 −12
Original line number Diff line number Diff line 
'''

Author:

    windz

Usage:

    snakemake -j 15 --cluster 'bsub -J {rulename} -n {threads} -o log/%J.stdout -e log/%J.stderr -R span[hosts=1]' -s flepseq.smk

'''



# create log dir

if not os.path.exists('log'):

    os.mkdir('log')





configfile: 'config.yml'

sample=config['sample']

genome_data=config['genome']
@@ -28,6 +21,7 @@ rule fastq_to_fasta: 
    output:

        'basecalled_data/{sample}.fasta'

    threads: 1

    conda: 'flepseq'

    shell:

        '''

python script/fastqdir2fasta.py --indir {input} --out {output}
@@ -42,6 +36,7 @@ rule mapping_to_genome: 
    params:

        genome=genome_data

    threads: 36

    conda: 'flepseq'

    shell:

        '''

minimap2 -t {threads} -ax splice --secondary=no -G 12000 {params.genome} {input} | samtools sort -@ {threads} -o {output.bam} -
@@ -56,6 +51,7 @@ rule find_3linker: 
    output:

        'aligned_data/{sample}.adapter.result.txt'

    threads: 36

    conda: 'flepseq'

    shell:

        '''

python script/adapterFinder.py --inbam {input.bam} --inseq {input.fasta} --out {output} --threads {threads} --mode 1
@@ -70,6 +66,7 @@ rule polyacaller: 
    output:

        'aligned_data/{sample}.polyA_tail.result.txt'

    threads: 36

    conda: 'flepseq'

    shell:

        '''

python script/PolyACaller.py --inadapter {input.adapter_result} --summary {input.sequencing_summary}  --fast5dir {input.fast5_dir} --out {output} --threads {threads}
@@ -84,6 +81,7 @@ rule identify_read_info: 
    params:

        'genome_data/exon_intron_pos.repr.bed'

    threads: 1

    conda: 'flepseq'

    shell:

        '''

python script/extract_read_info.py --inbed {params} --inbam {input} --out {output}
@@ -100,9 +98,9 @@ rule merge_info: 
    threads: 1

    params:

        'Nanopore'

    conda: 'flepseq'

    shell:

        '''

export PATH=/public/home/mowp/anaconda3/envs/R/bin:$PATH

Rscript script/merge_read_info.R --type {params} --inreadinfo {input.read_info} --inadapter {input.adapter} --inpolya {input.polya} --out {output.read_info_result}

        '''
@@ -118,10 +116,10 @@ rule splicing_kinetics: 
    params:

        inbed='genome_data/exon_intron_pos.repr.bed',

        select_intron='genome_data/select_introns.txt'

    conda: 'flepseq'

    shell:

        '''

python script/prepare_data_for_splice_kinetics.py --inreadinfo {input.read_info} --inbed {params.inbed} --out {output.splicing_data}

export PATH=/public/home/mowp/anaconda3/envs/R/bin:$PATH

Rscript script/plot_intron_splicing_kinetics.R --inrelpos {output.splicing_data} --inreadinfo {input.read_info} --inintron {params.select_intron} --out {output.splicing_kinetics} --pdf {output.figure}

        '''
@@ -135,9 +133,8 @@ rule intron_retention_ratio: 
        rna_ir='results/{sample}.read.rna.ir.stat',

        intron_ir='results/{sample}.read.intron.ir.stat',

    threads: 1

    conda: 'flepseq'

    shell:

        '''

export PATH=/public/home/mowp/anaconda3/envs/R/bin:$PATH

Rscript script/cal_polya_transcript_ir.R --inrelpos {input.splicing_data} --inreadinfo {input.read_info} --outrna {output.rna_ir} --outintron {output.intron_ir}

        '''

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