Hisat2 -Can it align paired readfiles, and unpaired read files leftover from trimming/QC simultaneously?
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2.5 years ago
RNAseqer ▴ 160

Hello all,

I am new to RNAseq and have just started working with HISAT2. Using trimmomatic I QCed an illumina paired end run and got 4 files: paired1.fq, paired2.fq were my paired reads after QC and unpaired1.fq, unpaired2.fq were files containing unpaired reads (presumably their partners didn't make the cut).

I went to run HISAT2 for the first time and tried to use the following commandline:

hisat2 -x genome_snp_tran -1 paired1.fq,-2 paired2.fq -U unpaired1.fq,unpaired2.fq -S test_output


only to get the following error:

Extra parameter(s) specified: "trimmed2paired.fastq" Error: Encountered internal HISAT2 exception (#1)

This problem is solved by deleting one set of reads (either the paired or unpaired files) at which point the program starts running. Can I take it from this that Hisat2 can only work with one type of data at a time? If so what is the best way to deal with my unpaired read file? Should I simply treat all the QCed reads as SE data and run it in unpaired mode? Or should I try to produce output for both the paired and unpaired files separately then combine the output somehow later? Or is there a way to use both paired and unpaired reads simultaneously with a correctly worded commandline. Is so could you provide an example?

Any help you could provide in this matter would be greatly appreciated!

rna-seq alignment HISAT2 Paired end • 1.3k views
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How many percent of the total reads are in unpaired? If it is a small amount, I would ignore them. In any case, do not treat the paired-end data as single-end, this would throw away information.

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The unpaired reads are 6.4% of the total.

Its really a non-issue for these particular reads since they are not the research data I hope to be working with, but rather published RNAseq data I downloaded to practice with. As I said, I'm new to RNAseq so I've created a flow chart/pipeline representing what I believe are the best practices and most sensitive/accurate/computationally efficient software. I'm just beginning to run through it to now.

That being said this sort of troubleshooting is exactly why I'm doing this. Thank you for your advice!

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The command-line you provided does not correspond to the error message, please copy the exact command-line used. Aldo, the command you pasted above has an extra comma between -1and -2

-1 paired1.fq,-2 paired2.fq

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Sorry, was trying to trim the fat of file names to make that commandline easier to read:

\$ hisat2 -x genome_snp_tran -1 trimmed1paired.fastq,-2 trimmed2paired.fastq -U trimmed1unpaired.fastq, trimmed2unpaired.fastq -S testoutput2

gives message:

Extra parameter(s) specified: "trimmed2paired.fastq", "trimmed2unpaired.fastq" Error: Encountered internal HISAT2 exception (#1) Command: /Users/mylapple/hisat2-2.1.0/hisat2-align-s --wrapper basic-0 -x genome_snp_tran -S testoutput2 -U trimmed1unpaired.fastq trimmed2paired.fastq trimmed2unpaired.fastq (ERR): hisat2-align exited with value 1