00.21-D-Apul-BS-genome
================
Sam White
2025-01-02

- <a href="#1-background" id="toc-1-background">1 Background</a>
- <a href="#2-inputs" id="toc-2-inputs">2 Inputs</a>
- <a href="#3-outputs" id="toc-3-outputs">3 Outputs</a>
- <a href="#4-create-a-bash-variables-file"
  id="toc-4-create-a-bash-variables-file">4 Create a Bash variables
  file</a>
- <a href="#5-bisfulite-conversion" id="toc-5-bisfulite-conversion">5
  Bisfulite conversion</a>
  - <a href="#51-inpect-bs-output" id="toc-51-inpect-bs-output">5.1 Inpect
    BS output</a>
  - <a href="#52-compress-output-folder"
    id="toc-52-compress-output-folder">5.2 Compress output folder</a>
  - <a href="#53-create-md5sum" id="toc-53-create-md5sum">5.3 Create
    MD5sum</a>
- <a href="#6-references" id="toc-6-references">6 REFERENCES</a>

# 1 Background

This Rmd file will create a bisulfite-converted genome by, and for,
Bismark (Krueger and Andrews 2011) using the `Apulchra-genome.fa` file.
The genome FastA was taken from the [deep-dive-expression
Wiki](https://github.com/urol-e5/deep-dive-expression/wiki/00-Genomic-Resources#acropora-pulchra)
(GitHub Wiki), but I’m not sure of its exact origin.

Due to large sizes of output files, the files cannot be sync’d to
GitHub. As such, the output directories will be gzipped and available
here:

- <https://gannet.fish.washington.edu/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome.tar.gz>
  (1.3GB)

- <https://gannet.fish.washington.edu/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome.tar.gz.md5>

  - MD5: `9e1db5875f210007a43e2083f01c2db9`

# 2 Inputs

- Directory containing a FastA file with the file extension: .fa or
  .fasta (also ending in .gz).

# 3 Outputs

- CT Conversion

  - Bowtie2 index files.
  - CT conversion FastA

- GA conversion

  - Bowtie2 index files.
  - GA conversion FastA.

# 4 Create a Bash variables file

This allows usage of Bash variables across R Markdown chunks.

``` bash
{
echo "#### Assign Variables ####"
echo ""

echo "# Data directories"
echo 'export timeseries_dir=/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular'
echo 'export output_dir_top=${timeseries_dir}/D-Apul/data'
echo 'export genome_dir=${timeseries_dir}/D-Apul/data'
echo ""

echo "# Paths to programs"
echo 'export programs_dir="/home/shared"'
echo 'export bismark_dir="${programs_dir}/Bismark-0.24.0"'
echo 'export bowtie2_dir="${programs_dir}/bowtie2-2.4.4-linux-x86_64"'
echo ""

echo "# Set number of CPUs to use"
echo 'export threads=20'
echo ""

echo "# Print formatting"
echo 'export line="--------------------------------------------------------"'
echo ""
} > .bashvars

cat .bashvars
```

    #### Assign Variables ####

    # Data directories
    export timeseries_dir=/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular
    export output_dir_top=${timeseries_dir}/D-Apul/data
    export genome_dir=${timeseries_dir}/D-Apul/data

    # Paths to programs
    export programs_dir="/home/shared"
    export bismark_dir="${programs_dir}/Bismark-0.24.0"
    export bowtie2_dir="${programs_dir}/bowtie2-2.4.4-linux-x86_64"

    # Set number of CPUs to use
    export threads=20

    # Print formatting
    export line="--------------------------------------------------------"

# 5 Bisfulite conversion

``` bash
# Load bash variables into memory
source .bashvars

${bismark_dir}/bismark_genome_preparation \
${genome_dir} \
--parallel ${threads} \
--bowtie2 \
--path_to_aligner ${bowtie2_dir} \
1> ${genome_dir}/Apulchra-bs-genome.stderr
```

    Using 20 threads for the top and bottom strand indexing processes each, so using 40 cores in total
    Writing bisulfite genomes out into a single MFA (multi FastA) file

    Bisulfite Genome Indexer version v0.24.0 (last modified: 19 May 2022)

    Step I - Prepare genome folders - completed



    Step II - Genome bisulfite conversions - completed


    Bismark Genome Preparation - Step III: Launching the Bowtie 2 indexer
    Building a SMALL index
    Building a SMALL index

    =========================================

    Parallel genome indexing complete. Enjoy!

## 5.1 Inpect BS output

``` bash
# Load bash variables into memory
source .bashvars

tree -h ${genome_dir}/Bisulfite_Genome
```

    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome
    ├── [4.0K]  CT_conversion
    │   ├── [169M]  BS_CT.1.bt2
    │   ├── [124M]  BS_CT.2.bt2
    │   ├── [1.5K]  BS_CT.3.bt2
    │   ├── [124M]  BS_CT.4.bt2
    │   ├── [169M]  BS_CT.rev.1.bt2
    │   ├── [124M]  BS_CT.rev.2.bt2
    │   └── [504M]  genome_mfa.CT_conversion.fa
    └── [4.0K]  GA_conversion
        ├── [169M]  BS_GA.1.bt2
        ├── [124M]  BS_GA.2.bt2
        ├── [1.5K]  BS_GA.3.bt2
        ├── [124M]  BS_GA.4.bt2
        ├── [169M]  BS_GA.rev.1.bt2
        ├── [124M]  BS_GA.rev.2.bt2
        └── [504M]  genome_mfa.GA_conversion.fa

    2 directories, 14 files

## 5.2 Compress output folder

``` bash
source .bashvars

tar -czvf ${genome_dir}/Bisulfite_Genome.tar.gz ${genome_dir}/Bisulfite_Genome
```

    tar: Removing leading `/' from member names
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/CT_conversion/
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/CT_conversion/BS_CT.4.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/CT_conversion/BS_CT.rev.1.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/CT_conversion/genome_mfa.CT_conversion.fa
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/CT_conversion/BS_CT.1.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/CT_conversion/BS_CT.2.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/CT_conversion/BS_CT.3.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/CT_conversion/BS_CT.rev.2.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/GA_conversion/
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/GA_conversion/BS_GA.rev.1.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/GA_conversion/BS_GA.2.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/GA_conversion/BS_GA.4.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/GA_conversion/genome_mfa.GA_conversion.fa
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/GA_conversion/BS_GA.rev.2.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/GA_conversion/BS_GA.1.bt2
    /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/Bisulfite_Genome/GA_conversion/BS_GA.3.bt2

## 5.3 Create MD5sum

``` bash
source .bashvars

cd ${genome_dir}

md5sum Bisulfite_Genome.tar.gz | tee Bisulfite_Genome.tar.gz.md5
```

    399a196fc1dfc202a9f084a8fb342dd9  Bisulfite_Genome.tar.gz

# 6 REFERENCES

<div id="refs" class="references csl-bib-body hanging-indent">

<div id="ref-krueger2011" class="csl-entry">

Krueger, Felix, and Simon R. Andrews. 2011. “Bismark: A Flexible Aligner
and Methylation Caller for Bisulfite-Seq Applications.” *Bioinformatics*
27 (11): 1571–72. <https://doi.org/10.1093/bioinformatics/btr167>.

</div>

</div>