Genome Indexing - P.tuahiniensis Using Hisat2 on Raven

2025
Pocillopora tuahiniensis
E5
coral
Hisat2
Author

Sam White

Published

March 7, 2025

INTRO

In preparation for RNA-seq analysis for urol-e5/timeseries_molecular (GitHub repo) project, we needed to index the genome using HISAT2. I had previously fixed the GFF and prepared a corresponding GTF on 20250114 so that we could identify exons splice sites - both of which are useful during genome indexing to identify alternative isoforms in downstream applications.

Note

The contents below are from markdown knitted from 02.10-F-Ptua-RNAseq-genome-index-HiSat2.Rmd (commit 81c1117).

1 Background

This notebook will build an index of the P.tuahiniensis genome using HISAT2 (Kim et al. 2019). It utilizes the GTF file created in 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:

2 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 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

# 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

# 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

# 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

# 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

References

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.