Question: Hello,

I have been strugelling to run Hisat2 code for alignment of several fastq files from human
I have read as many post as I could online and I went through the protocol here
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032908/

At the moment, i have the latest binary version of hisat2 in $HOME/bin
When I do hisat2 in the terminal and shows that I don't import any input but it indicates that it is there and can be invoked

No index, query, or output file specified!
HISAT2 version 2.1.0 by Daehwan Kim (infphilo@gmail.com, www.ccb.jhu.edu/people/infphilo)
Usage:

I have 10 fasta files on a folder named data on my desktop.
I have put the three scripts attached to the protocl in that folder and the files are shown below

NIHMS816842-supplement-README.txt
RNAseq_shell_script.sh
SRR1_1.fastq.gz	SRR9_1.fastq.gz
SRR1_2.fastq.gz	SRR9_2.fastq.gz
SRR2_1.fastq.gz	SRR10_1.fastq.gz
SRR2_2.fastq.gz	SRR10_2.fastq.gz
SRR3_1.fastq.gz
SRR3_2.fastq.gz
SRR4_1.fastq.gz
SRR4_2.fastq.gz
SRR5_1.fastq.gz
SRR5_2.fastq.gz
SRR6_1.fastq.gz
SRR6_2.fastq.gz
SRR7_1.fastq.gz
SRR7_2.fastq.gz	rnaseq_ballgown.R
SRR8_1.fastq.gz	rnaseq_pipeline.config.sh
SRR8_2.fastq.gz

Can you please let me know how I can run the code to align these fastq files with a Human gene reference ?

I did not change anything on any of the script which I believe that I must change the one in rnaseq_pipline.config.sh

At this moment, it is

NUMCPUS=8
HISAT2=$(which hisat2)
STRINGTIE=$(which stringtie)
SAMTOOLS=$(which samtools)
BASEDIR=$(pwd -P)/chrX_data
FASTQLOC="$BASEDIR/samples"
GENOMEIDX="$BASEDIR/indexes/chrX_tran"
GTFFILE="$BASEDIR/genes/chrX.gtf"
PHENODATA="$BASEDIR/geuvadis_phenodata.csv"
TEMPLOC="./tmp" #this will be relative to the output directory
reads1=(${FASTQLOC}/*_1.*)
reads1=("${reads1[@]##*/}")
reads2=("${reads1[@]/_1./_2.}")

All what I want is to get the htseq-count. txt files for each sample

based on this post https://www.biostars.org/p/223404/ If I do the following

for f in `ls -1 *_1.fastq.gz | sed 's/_1.fastq.gz//' `
do
echo hisat2 -x mm10idx -1 ${f}_1.fastq.gz -2 ${f}_2.fastq.gz -S ${f}.bam
done

It will generate all the bam files but all empty. if I echo the do , it will be the output as an example

hisat2 -x mm10idx -1 SRR1_1.fastq.gz -2 SRR1_2.fastq.gz -S SRR1.bam
hisat2 -x mm10idx -1 SRR2_1.fastq.gz -2 SRR42_2.fastq.gz -S SRR2.bam
hisat2 -x mm10idx -1 SRR3_1.fastq.gz -2 SRR3_2.fastq.gz -S SRR3.bam
hisat2 -x mm10idx -1 SRR4_1.fastq.gz -2 SRR_2.fastq.gz -S SRR4.bam
hisat2 -x mm10idx -1 SRR5_1.fastq.gz -2 SRR5_2.fastq.gz -S SRR5.bam
hisat2 -x mm10idx -1 SRR6_1.fastq.gz -2 SRR6_2.fastq.gz -S SRR6.bam
hisat2 -x mm10idx -1 SRR7_1.fastq.gz -2 SRR7_2.fastq.gz -S SRR7.bam
hisat2 -x mm10idx -1 SRR8_1.fastq.gz -2 SRR8_2.fastq.gz -S SRR8.bam
hisat2 -x mm10idx -1 SRR9_1.fastq.gz -2 SRR9_2.fastq.gz -S SRR9.bam
hisat2 -x mm10idx -1 SRR10_1.fastq.gz -2 SRR10_2.fastq.gz -S SRR10.bam

On the other hand, where should I involve the reference genome? for example, I downloaded the hg19 from here https://ccb.jhu.edu/software/hisat2/index.shtml

Answer: You're trying to do too many things at once, which is just going to cause confusion for someone new to the field. Start out with a single sample that manually type the command for. Once that works properly you can move on to multiple samples in a loop.

Since you apparently downloaded the hg19 prebuilt reference, you need to specify where it is with the `-x` option. In your examples, that was `-x mm10idx`, but presumably something like `-x /some/path/to/the/downloaded/files/hg19` would make more sense.