02.10-F-Ptua-RNAseq-genome-index-HiSat2
================
Sam White
2025-03-06

- <a href="#1-background" id="toc-1-background">1 Background</a>
- <a href="#2-create-a-bash-variables-file"
  id="toc-2-create-a-bash-variables-file">2 Create a Bash variables
  file</a>
- <a href="#3-identify-exons" id="toc-3-identify-exons">3 Identify
  exons</a>
- <a href="#4-identify-splice-sites" id="toc-4-identify-splice-sites">4
  Identify splice sites</a>
- <a href="#5-build-hisat2-genome-index"
  id="toc-5-build-hisat2-genome-index">5 Build HISAT2 genome index</a>
- <a href="#6-list-hisat2-index-files"
  id="toc-6-list-hisat2-index-files">6 List Hisat2 index files</a>

# 1 Background

This notebook will build an index of the *P.tuahiniensis* genome using
[HISAT2](https://github.com/DaehwanKimLab/hisat2) (Kim et al. 2019). It
utilizes the GTF file created in
[`00-genome-GFF-formatting.Rmd`](https://github.com/urol-e5/timeseries_molecular/blob/e4361d794b8a6887bc80a979491cb931e93f3e2a/F-Ptua/code/00.00-genome-GFF-formatting.Rmd).

Due to the large file sizes of the outputs HISAT2 index files (`*.ht2`),
they cannot be added to GitHub. As such, they are available for download
from here:

- <https://gannet.fish.washington.edu/gitrepos/urol-e5/timeseries_molecular/F-Ptua/output/02.10-F-Ptua-RNAseq-genome-index-HiSat2>

# 2 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 genome_dir="${timeseries_dir}/F-Ptua/data"'
echo 'export output_dir_top=${timeseries_dir}/F-Ptua/output/02.10-F-Ptua-RNAseq-genome-index-HiSat2'
echo ""

echo "# Input/output files"
echo 'export genome_index_name="Pocillopora_meandrina_HIv1.assembly"'
echo 'export exons="${output_dir_top}/Pocillopora_meandrina_HIv1.assembly_hisat2_exons.tab"'
echo 'export genome_gff="${genome_dir}/Pocillopora_meandrina_HIv1.genes-validated.gff3"'
echo 'export genome_fasta="${genome_dir}/Pocillopora_meandrina_HIv1.assembly.fasta"'
echo 'export splice_sites="${output_dir_top}/Pocillopora_meandrina_HIv1.assembly_hisat2_splice_sites.tab"'
echo 'export transcripts_gtf="${genome_dir}/Pocillopora_meandrina_HIv1.genes-validated.gtf"'

echo "# Paths to programs"
echo 'export programs_dir="/home/shared"'
echo 'export hisat2_dir="${programs_dir}/hisat2-2.2.1"'
echo ""
echo 'export hisat2_build="${hisat2_dir}/hisat2-build"'
echo 'export hisat2_exons="${hisat2_dir}/hisat2_extract_exons.py"'
echo 'export hisat2_splice_sites="${hisat2_dir}/hisat2_extract_splice_sites.py"'
echo ""

echo "# Set number of CPUs to use"
echo 'export threads=40'
echo ""

echo "# Programs associative array"
echo "declare -A programs_array"
echo "programs_array=("
echo '[hisat2]="${hisat2}" \'
echo '[hisat2_build]="${hisat2_build}" \'
echo '[hisat2_exons]="${hisat2_exons}" \'
echo '[hisat2_splice_sites]="${hisat2_splice_sites}" \'
echo ")"
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 genome_dir="${timeseries_dir}/F-Ptua/data"
    export output_dir_top=${timeseries_dir}/F-Ptua/output/02.10-F-Ptua-RNAseq-genome-index-HiSat2

    # Input/output files
    export genome_index_name="Pocillopora_meandrina_HIv1.assembly"
    export exons="${output_dir_top}/Pocillopora_meandrina_HIv1.assembly_hisat2_exons.tab"
    export genome_gff="${genome_dir}/Pocillopora_meandrina_HIv1.genes-validated.gff3"
    export genome_fasta="${genome_dir}/Pocillopora_meandrina_HIv1.assembly.fasta"
    export splice_sites="${output_dir_top}/Pocillopora_meandrina_HIv1.assembly_hisat2_splice_sites.tab"
    export transcripts_gtf="${genome_dir}/Pocillopora_meandrina_HIv1.genes-validated.gtf"
    # Paths to programs
    export programs_dir="/home/shared"
    export hisat2_dir="${programs_dir}/hisat2-2.2.1"

    export hisat2_build="${hisat2_dir}/hisat2-build"
    export hisat2_exons="${hisat2_dir}/hisat2_extract_exons.py"
    export hisat2_splice_sites="${hisat2_dir}/hisat2_extract_splice_sites.py"

    # Set number of CPUs to use
    export threads=40

    # Programs associative array
    declare -A programs_array
    programs_array=(
    [hisat2]="${hisat2}" \
    [hisat2_build]="${hisat2_build}" \
    [hisat2_exons]="${hisat2_exons}" \
    [hisat2_splice_sites]="${hisat2_splice_sites}" \
    )

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

# 3 Identify exons

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

# Make directories, if they don't exist
mkdir --parents "${output_dir_top}"

# Create Hisat2 exons tab file
"${programs_array[hisat2_exons]}" \
"${transcripts_gtf}" \
> "${exons}"

head "${exons}"
```

    Pocillopora_meandrina_HIv1___Sc0000000  10770   11116   +
    Pocillopora_meandrina_HIv1___Sc0000000  12783   12874   +
    Pocillopora_meandrina_HIv1___Sc0000000  13539   13642   +
    Pocillopora_meandrina_HIv1___Sc0000000  14319   14391   +
    Pocillopora_meandrina_HIv1___Sc0000000  14676   14751   +
    Pocillopora_meandrina_HIv1___Sc0000000  15025   15130   +
    Pocillopora_meandrina_HIv1___Sc0000000  15459   15574   +
    Pocillopora_meandrina_HIv1___Sc0000000  17212   17286   +
    Pocillopora_meandrina_HIv1___Sc0000000  17877   17994   +
    Pocillopora_meandrina_HIv1___Sc0000000  18563   18696   +

# 4 Identify splice sites

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

# Create Hisat2 splice sites tab file
"${programs_array[hisat2_splice_sites]}" \
"${transcripts_gtf}" \
> "${splice_sites}"

head "${splice_sites}"
```

    Pocillopora_meandrina_HIv1___Sc0000000  11116   12783   +
    Pocillopora_meandrina_HIv1___Sc0000000  12874   13539   +
    Pocillopora_meandrina_HIv1___Sc0000000  13642   14319   +
    Pocillopora_meandrina_HIv1___Sc0000000  14391   14676   +
    Pocillopora_meandrina_HIv1___Sc0000000  14751   15025   +
    Pocillopora_meandrina_HIv1___Sc0000000  15130   15459   +
    Pocillopora_meandrina_HIv1___Sc0000000  15574   17212   +
    Pocillopora_meandrina_HIv1___Sc0000000  17286   17877   +
    Pocillopora_meandrina_HIv1___Sc0000000  17994   18563   +
    Pocillopora_meandrina_HIv1___Sc0000000  18696   19169   +

# 5 Build HISAT2 genome index

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

# Change to working directory
cd "${output_dir_top}"

# Build Hisat2 reference index using splice sites and exons
"${programs_array[hisat2_build]}" \
"${genome_fasta}" \
"${genome_index_name}" \
--exon "${exons}" \
--ss "${splice_sites}" \
-p "${threads}" \
2> "${genome_index_name}"-hisat2_build.err

ls -lh
```

    total 773M
    -rw-r--r-- 1 sam sam 227M Mar  6 17:18 Pocillopora_meandrina_HIv1.assembly.1.ht2
    -rw-r--r-- 1 sam sam  91M Mar  6 17:18 Pocillopora_meandrina_HIv1.assembly.2.ht2
    -rw-r--r-- 1 sam sam 1.9K Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly.3.ht2
    -rw-r--r-- 1 sam sam  90M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly.4.ht2
    -rw-r--r-- 1 sam sam 245M Mar  6 17:19 Pocillopora_meandrina_HIv1.assembly.5.ht2
    -rw-r--r-- 1 sam sam  92M Mar  6 17:19 Pocillopora_meandrina_HIv1.assembly.6.ht2
    -rw-r--r-- 1 sam sam 7.4M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly.7.ht2
    -rw-r--r-- 1 sam sam 1.5M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly.8.ht2
    -rw-r--r-- 1 sam sam  15K Mar  6 17:19 Pocillopora_meandrina_HIv1.assembly-hisat2_build.err
    -rw-r--r-- 1 sam sam  12M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly_hisat2_exons.tab
    -rw-r--r-- 1 sam sam 9.7M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly_hisat2_splice_sites.tab

# 6 List Hisat2 index files

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

for index in "${output_dir_top}"/*.ht2
do
  cp ${index} ${genome_dir}
done

ls -lh "${output_dir_top}"
```

    total 773M
    -rw-r--r-- 1 sam sam 227M Mar  6 17:18 Pocillopora_meandrina_HIv1.assembly.1.ht2
    -rw-r--r-- 1 sam sam  91M Mar  6 17:18 Pocillopora_meandrina_HIv1.assembly.2.ht2
    -rw-r--r-- 1 sam sam 1.9K Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly.3.ht2
    -rw-r--r-- 1 sam sam  90M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly.4.ht2
    -rw-r--r-- 1 sam sam 245M Mar  6 17:19 Pocillopora_meandrina_HIv1.assembly.5.ht2
    -rw-r--r-- 1 sam sam  92M Mar  6 17:19 Pocillopora_meandrina_HIv1.assembly.6.ht2
    -rw-r--r-- 1 sam sam 7.4M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly.7.ht2
    -rw-r--r-- 1 sam sam 1.5M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly.8.ht2
    -rw-r--r-- 1 sam sam  15K Mar  6 17:19 Pocillopora_meandrina_HIv1.assembly-hisat2_build.err
    -rw-r--r-- 1 sam sam  12M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly_hisat2_exons.tab
    -rw-r--r-- 1 sam sam 9.7M Mar  6 17:12 Pocillopora_meandrina_HIv1.assembly_hisat2_splice_sites.tab

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

<div id="ref-kim2019" class="csl-entry">

Kim, Daehwan, Joseph M. Paggi, Chanhee Park, Christopher Bennett, and
Steven L. Salzberg. 2019. “Graph-Based Genome Alignment and Genotyping
with HISAT2 and HISAT-Genotype.” *Nature Biotechnology* 37 (8): 907–15.
<https://doi.org/10.1038/s41587-019-0201-4>.

</div>

</div>