INTRO
In preparation for Bismark alignments, we needed to generate a bisulfite-converted genome for P.tuahiniensis, as one did not already exist.
The resulting output files were added to the Genomic Resources Handbook page and the timeseries_molecular repo Wiki.
Note
The contents below are from markdown knitted from 00.21-F-Ptua-BS-genome.Rmd (commit 7b0bb87).
1 Background
This Rmd file will create a bisulfite-converted genome by, and for, Bismark (Krueger and Andrews 2011) using the Pocillopora_meandrina_HIv1.assembly.fasta file. The genome FastA was taken from the Cyanophora webpage.
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:
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.
{
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=${timeseries_dir}/F-Ptua/data'
echo 'export genome_dir=${timeseries_dir}/F-Ptua/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=${timeseries_dir}/F-Ptua/data
export genome_dir=${timeseries_dir}/F-Ptua/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
# 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}/Ptua-bs-genome.stderrUsing 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
# Load bash variables into memory
source .bashvars
tree -h ${genome_dir}/Bisulfite_Genome/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome
├── [4.0K] CT_conversion
│ ├── [124M] BS_CT.1.bt2
│ ├── [ 90M] BS_CT.2.bt2
│ ├── [1.9K] BS_CT.3.bt2
│ ├── [ 90M] BS_CT.4.bt2
│ ├── [124M] BS_CT.rev.1.bt2
│ ├── [ 90M] BS_CT.rev.2.bt2
│ └── [359M] genome_mfa.CT_conversion.fa
└── [4.0K] GA_conversion
├── [124M] BS_GA.1.bt2
├── [ 90M] BS_GA.2.bt2
├── [1.9K] BS_GA.3.bt2
├── [ 90M] BS_GA.4.bt2
├── [124M] BS_GA.rev.1.bt2
├── [ 90M] BS_GA.rev.2.bt2
└── [359M] genome_mfa.GA_conversion.fa
2 directories, 14 files5.2 Compress output folder
source .bashvars
tar -czvf ${genome_dir}/Bisulfite_Genome.tar.gz ${genome_dir}/Bisulfite_Genometar: Removing leading `/' from member names
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/CT_conversion/
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/CT_conversion/BS_CT.4.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/CT_conversion/BS_CT.rev.1.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/CT_conversion/genome_mfa.CT_conversion.fa
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/CT_conversion/BS_CT.1.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/CT_conversion/BS_CT.2.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/CT_conversion/BS_CT.3.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/CT_conversion/BS_CT.rev.2.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/GA_conversion/
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/GA_conversion/BS_GA.rev.1.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/GA_conversion/BS_GA.2.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/GA_conversion/BS_GA.4.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/GA_conversion/genome_mfa.GA_conversion.fa
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/GA_conversion/BS_GA.rev.2.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/GA_conversion/BS_GA.1.bt2
/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/F-Ptua/data/Bisulfite_Genome/GA_conversion/BS_GA.3.bt25.3 Create MD5sum
source .bashvars
cd ${genome_dir}
md5sum Bisulfite_Genome.tar.gz | tee Bisulfite_Genome.tar.gz.md53cef8ba7316aa2bb15eafbbb438470f2 Bisulfite_Genome.tar.gz6 REFERENCES
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.