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HISAT2 is a fast  transcriptome-aware mapper that is part of the new tuxedo suite of tools. These tools start with raw fastq files and produce genes, gene counts and identifies differentially expressed genes. HISAT2 uses a global, whole-genome index and tens of thousands of small local indexes to perform mapping in an extremely fast manner.

 HISAT2, like BWA and bowtie, uses burrows-wheeler transform (BWT) to compress genomes such that they require very little memory to store. Like BWA and bowtie, it builds indexes out of the transformed genomes using a special scheme called FM indexing. This makes it possible to search through these genomes rapidly. Unlike BWA and bowtie, HISAT2 builds a whole genome global index and thousands of small local indexes (both using the BWT/FM methods) to make spliced alignment possible.

Run HISAT2

First, make sure you are in the right directory for this exercise.

cds
cd my_rnaseq_course
cd day_1_partB/hisat_exercise
ls

Next, see if HISAT2 is a module that is available on stampede.

module spider hisat

As you can see, HISAT2 is not currently a module on stampede.  When a program is not available on stampede, you can install it locally in your home or work directory.  I have already installed HISAT in my work directory and since my work directory is in your path, you should be able to run hisat2.

Part 1. Create a index of your reference

NO NEED TO RUN THIS NOW- YOUR INDEX HAS ALREADY BEEN BUILT!

hisat2-build reference/genome.fa reference/genome.fa

Part 2. Align the samples to reference using hisat2

nano commands.hisat2

hisat2 -x reference/genome.fa -1 data/GSM794483_C1_R1_1.fq -2 data/GSM794483_C1_R1_2.fq -S GSM794483_C1.sam --phred33 --novel-splicesite-outfile GSM794483_C1.junctions --rna-strandness RF --dta -t
hisat2 -x reference/genome.fa -1 data/GSM794484_C1_R2_1.fq -2 data/GSM794484_C1_R2_2.fq -S GSM794484_C1.sam --phred33 --novel-splicesite-outfile GSM794484_C1.junctions --rna-strandness RF --dta -t
hisat2 -x reference/genome.fa -1 data/GSM794485_C1_R3_1.fq -2 data/GSM794485_C1_R3_2.fq -S GSM794485_C1.sam --phred33 --novel-splicesite-outfile GSM794485_C1.junctions --rna-strandness RF --dta -t
hisat2 -x reference/genome.fa -1 data/GSM794486_C2_R1_1.fq -2 data/GSM794486_C2_R1_2.fq -S GSM794486_C1.sam --phred33 --novel-splicesite-outfile GSM794486_C1.junctions --rna-strandness RF --dta -t
hisat2 -x reference/genome.fa -1 data/GSM794487_C2_R2_1.fq -2 data/GSM794487_C2_R2_2.fq -S GSM794487_C1.sam --phred33 --novel-splicesite outfile GSM794487_C1.junctions --rna-strandness RF --dta -t
hisat2 -x reference/genome.fa -1 data/GSM794488_C2_R3_1.fq -2 data/GSM794488_C2_R3_2.fq -S GSM794488_C1.sam --phred33 --novel-splicesite-outfile GSM794488_C1.junctions --rna-strandness RF --dta -t