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Old 03-03-2019, 09:31 AM   #1
frankenndoc
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Default Issue with Hisat2

Hello,
Iím trying to use Hisat2 to align human RNA-seq reads. However I always get the following error (see below). Iím using the GRC38 genome_tran index from the Hisat2 website, running hisat2 on 8 cores. I don't understand what this means, did the run complete successfully?

40321622 reads; of these:
40321622 (100.00%) were unpaired; of these:
2059219 (5.11%) aligned 0 times
31974150 (79.30%) aligned exactly 1 time
6288253 (15.60%) aligned >1 times
94.89% overall alignment rate
Warning: Could not open read file "/home/RNA-seq/Human/eynden/clb-bar-ctrl.fastq" for reading; skipping...
Error: No input read files were valid
(ERR): hisat2-align exited with value 1
Error while flushing and closing outputError while flushing and closing output
Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing outputterminate called recursively
Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing outputError while flushing and closing output

Error while flushing and closing outputterminate called after throwing an instance of Ďint'
Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing output
terminate called after throwing an instance of 'int'
Error while flushing and closing output

Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing output
Error while flushing and closing output
terminate called after throwing an instance of 'int'
Error while flushing and closing output
terminate called recursively
terminate called recursively
terminate called recursively
terminate called recursively
terminate called recursively
terminate called recursively
terminate called recursively
terminate called recursively
Aborted (core dumped)
(ERR): hisat2-align exited with value 134
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Old 03-03-2019, 12:35 PM   #2
GenoMax
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Are you trying to pass more than one set of files in your command line? Can you post your command line?
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Old 03-03-2019, 01:10 PM   #3
frankenndoc
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No, just the one file.
here's the command.
hisat2 -p 8 --dta -x ~/Databases/grch38_tran/genome_tran -U ~/RNA-seq/Human/eynden/clb-ge-ctrl.fastq -S ~/RNA-seq/Human/eynden/SAM/clb-ge-ctrl.sam
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Old 03-03-2019, 04:22 PM   #4
GenoMax
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That is odd because the error says that hisat is unable to read this file.

Code:
Warning: Could not open read file "/home/RNA-seq/Human/eynden/clb-bar-ctrl.fastq" for reading; skipping...
Error: No input read files were valid
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Old 03-03-2019, 04:31 PM   #5
frankenndoc
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I know, and before that it seems like it's doing the alignment too. There is a SAM file created but with this error I'm not sure if it's complete.
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Old 03-03-2019, 09:34 PM   #6
frankenndoc
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I just ran them again and it seemed to work for 4 datasets and then crashed on the 5th. Could it be a disk space issue? I logged on through remote desktop and there was a disk-space alert, though I still had around 30Gb free. wondering if Hisat2 makes some crazy large temp files or something while running that made it crash on this last run?
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Old 03-04-2019, 04:18 AM   #7
GenoMax
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Are your data files very large? If the SAM file being made gets very large then you could possibly run into a space issue.
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Old 03-04-2019, 05:47 AM   #8
frankenndoc
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Fasta files are around 6-10Gb, SAM files are about 10-15Gb for the successful runs. So I still have enough free disk space to handle one more but it just crashes when it reaches 30Gb of free disk space.
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Old 03-04-2019, 05:53 AM   #9
GenoMax
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Are you running this on a cluster/remote server? Perhaps your process is encountering disk quota limit?
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Old 03-04-2019, 06:16 AM   #10
frankenndoc
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that's possible, it's a remote machine, but I own it and I'm the only one using it. I'll check with my system admin, but is it routine for them to place limits?
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