6. Spliced Alignment to Reference Genome with HISAT2

In eukaryotic transcriptomes, mature messenger RNAs originate from genomic exons separated by introns that span from hundreds to hundreds of thousands of base pairs. Standard DNA mappers (BWA-MEM, Bowtie2) cannot align spliced RNA-seq reads because they penalize large gaps. Spliced aligners like HISAT2 (Hierarchical Indexing for Spliced Alignment of Transcripts) utilize a Hierarchical Graph FM-index (HGFM) to map split-reads across intron junctions with extreme speed and low memory (~4 GB RAM for human).

Building the Spliced HISAT2 Genome Index

# 1. Extract splice sites and exons from Ensembl/GENCODE GTF annotation
hisat2_extract_splice_sites.py ref/Homo_sapiens.GRCh38.110.gtf > ref/splice_sites.txt
hisat2_extract_exons.py ref/Homo_sapiens.GRCh38.110.gtf > ref/exons.txt

# 2. Build HISAT2 index incorporating annotated junctions
hisat2-build -p 16 \
  --ss ref/splice_sites.txt \
  --exon ref/exons.txt \
  ref/Homo_sapiens.GRCh38.dna.primary_assembly.fa \
  ref/hisat2_grch38_index

Executing Spliced Alignment with Samtools Streaming

# Spliced paired-end alignment piped directly into coordinate-sorted BAM
hisat2 -p 16 \
  --dta \
  -x ref/hisat2_grch38_index \
  -1 02_trimmed_data/Sample1_R1.trimmed.fastq.gz \
  -2 02_trimmed_data/Sample1_R2.trimmed.fastq.gz \
  --rna-strandness RF \
  --summary-file 04_alignment/Sample1_hisat2_summary.txt | \
  samtools view -@ 8 -bS - | \
  samtools sort -@ 8 -o 04_alignment/Sample1.sorted.bam -

# Index the BAM file
samtools index 04_alignment/Sample1.sorted.bam