Solve the problem of Hisat2:Align reads to genome

Question

[Kanoo]

Firstly, I set an index through Hisat2. When I use Hisat2 to alignment, there appears an error--

Error:could not open alignment output file.

But I have set an output file already, and it's properties: drwxrwxr-x.

The following is my attempt to eliminate the error：

1. I think maybe the folder is damaged:I rebulid a new folder to do alignment,still failed.--same error

2. I think maybe my index has something wrong: My friends of the same laboratory set index by hisat2 by them own,after comparing with my index,I found that there is no problem with my index.

3. Maybe this software can't handle too large files?(After QC,one sample of mine have 2 strand,named:R1.clean.fastq and R2.clean.fastq.each of the strand as big as 8Gb--one sample is 16Gb，105337344 rows.)So I cut 1/2 of R1 and R2,then use hisat2,but failed(same error). Then I try to cut head of 1/192 of R1(548632 rows of data）and R2(548632 rows of data） to use Hisat2,still failed.

head -n 548632 R1.clean.fastq

4. I think maybe some of the previous data are wrong, so I took a part from the very end of the data, took a piece of data from any part of the middle, and use hisat2,did it separately--failed(error).

tail -n 100000 R1.clean.fastq        sed -n '10000-20000p' R1.clean.fastq

5. Because yesterday I took out the first 50 rows from one of my samples and created a new folder to do hisat2,and suceed,today I take out 40 rows from yesterday samples,creat a new file to do hisat2,still failed.Why I can't do hisat2 on 40 rows that I took less than the sample yesterday？

6. I set same folder as yesterday,do the same work,and succeed.

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Finally, I proposed (find) that the output folder cannot be in the same folder as the sample file (that is, the input file), nor can it be a parallel folder with the sample file.

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Edit (MD) -- Note: this is most likely not true.

The output folder must be built under a folder that does not contain samples.

For example:

Wrong--input:/home/liquorice/QCsamples/xxx    output:/home/liquorice/Hisat2Result/xxx.sam
Wrong--input:/home/liquorice/QCsamples/xxx    output:/home/liquorice/QCsamples/Hisat2Result/xxx.sam
Right--input:/home/liquorice/QCsamples/xxx      output:/home/Hisat2Result/xxx.sam

hisat2 -x Hisat2index -1 /home/liquorice/liquoriceTrimResult/CT18TN1/R1.clean.fastq -2 /home/liquorice/liquoriceTrimResult/CT18TN1/R2.clean.fastq -S /home/try/Result/CT18TN1.sam 

Wish it helpful.

[ATpoint]

Hi Kanoo, this seems to be an issue with your system configuration. I can assure you hisat2 can handle files of arbitrary size and given read/write permissions are set properly, you can save input and output files at any location, no matter where the input files are.

[Kanoo]

Ok,I will check out.Thanks!

Answer 1

[Michael]

It looks like you are experiencing a Unix permission problem. You do not have write permissions in /home/liquorice/ but seemingly you have write permissions on /home/Hisat2Result/. Please check out these permissions.

touch /home/liquorice/testfile # error, permission denied
touch /home/Hisat2Result/testfile # should work
echo $HOME

These problems will be avoided if you avoid working across home directories (you are either user licorice or Hisat2Results and you should not write to another home directories) in the future. Your conclusion might be confusing, so I will edit it.