Question: Hisat2 -Can it align paired readfiles, and unpaired read files leftover from trimming/QC simultaneously?
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9 months ago by
RNAseqer 110
RNAseqer 110 wrote:

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!

ADD COMMENTlink modified 9 months ago by h.mon28k • written 9 months ago by RNAseqer 110

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.

ADD REPLYlink written 9 months ago by ATpoint26k

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!

ADD REPLYlink modified 9 months ago • written 9 months ago by RNAseqer 110

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
ADD REPLYlink modified 9 months ago • written 9 months ago by h.mon28k

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

ADD REPLYlink written 9 months ago by RNAseqer 110
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