Building Genome and Transcriptome Indexes for Ostreococcus tauri with HISAT2

Introduction

Ostreococcus tauri is a small eukaryotic organism with a relatively small genome, making it suitable for quick construction of genome and transcriptome indexes. In this README, we will outline the steps to build these indexes.

Prerequisites

• Files: Ostreococcus tauri genome file (.fna) and general transfer format file (.gtf)
• Software: Hisat2

Building the Genome Index

1. Run the following command to download the genomic fasta file.

wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/214/015/GCF_000214015.3_version_140606/GCF_000214015.3_version_140606_genomic.fna.gz

2. Run the following command to unzip the genomic fasta file.

gunzip GCF_000214015.3_version_140606_genomic.fna.gz

3. Run the following command to create a genome index folder.

mkdir GenomeIndex

4. Run the following command to build the genome index (4 threads used). Take a look in the GenomeIndex folder after executing this command.

hisat2-build GCF_000214015.3_version_140606_genomic.fna GenomeIndex/genome -p 4

Building the Transcriptome Index

1. Run the following command to download the general transfer file.

wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/214/015/GCF_000214015.3_version_140606/GCF_000214015.3_version_140606_genomic.gtf.gz

2. Run the following command to unzip the general transfer file.

gunzip GCF_000214015.3_version_140606_genomic.gtf.gz

3. Run the following command to create a transcriptome index folder.

mkdir TranscriptomeIndex

4. Run the following command to extract the splice sites from the Ostreococcus tauri genome (information about splice sites can be found in the .gtf file). Take a look in the TranscriptomeIndex folder after executing this command.

hisat2_extract_splice_sites.py GCF_000214015.3_version_140606_genomic.gtf > TranscriptomeIndex/SpliceSites.ss

5. Run the following command to extract the exons from the Ostreococcus tauri genome (information about exons can be found in the .gtf file). Take a look in the TranscriptomeIndex folder after executing this command.

hisat2_extract_exons.py GCF_000214015.3_version_140606_genomic.gtf > TranscriptomeIndex/Exons.exon 

6. Run the following command to build the transcriptome index (4 threads used). Take a look in the TranscriptomeIndex folder after executing this command.

hisat2-build --ss TranscriptomeIndex/SpliceSites.ss --exon TranscriptomeIndex/Exons.exon GCF_000214015.3_version_140606_genomic.fna TranscriptomeIndex/transcriptome -p 4 

Testing the Genome and Transcriptome Index

1. Run the following command to download a few spots from a sample record that is linked to Ostreococcus tauri.

fastq-dump --gzip --split-3 -X 1000 SRR7121135 

2. Run the following command to create a results folder

mkdir Results 

3. Align the RNA-seq reads to the genome index:

hisat2 -x GenomeIndex/genome -U SRR7121135_1.fastq.gz -S Results/SRR7121135_1_genome.sam 

4. Align the RNA-seq reads to the transcriptome index:

hisat2 -x TranscriptomeIndex/transcriptome -U SRR7121135_1.fastq.gz -S Results/SRR7121135_1_transcriptome.sam