RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

Indexing


RNA-seq_Flowchart


Create a HISAT2 index

Create a HISAT2 index for chr22 and the ERCC spike-in sequences. HISAT2 can incorporate exons and splice sites into the index file for alignment. First create a splice site file, then an exon file. Finally make the aligner FM index.

To learn more about how the HISAT2 indexing strategy is distinct from other next gen aligners refer to the HISAT publication.

    cd $RNA_HOME
    hisat2_extract_splice_sites.py $RNA_REF_GTF > $RNA_REFS_DIR/splicesites.tsv
    hisat2_extract_exons.py $RNA_REF_GTF > $RNA_REFS_DIR/exons.tsv
    hisat2-build -p 8 --ss $RNA_REFS_DIR/splicesites.tsv --exon $RNA_REFS_DIR/exons.tsv $RNA_REF_FASTA $RNA_REF_INDEX

[OPTIONAL] To create an index for all chromosomes instead of just chr22 you would do the following:

NOTE: The below example does NOT take advantage of adding the splice sites and exons to the index. If desired, you would make those files using the full GTF and add them to the command using the appropriate options.

WARNING: In order to index the entire human genome, HISAT2 requires 160GB of RAM. Your AWS instance size will run out of RAM.

    #cd /home/ubuntu/workspace/data/fasta/GRCh38
    #hisat2-build -p 8 Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa Homo_sapiens.GRCh38.dna_sm.primary_assembly