Question: bowtie "Saw ASCII character -54 but expected 33-based Phred qual." after -Q 33 fastx reverse complement
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gravatar for gufernandez10
2.1 years ago by
Chile
gufernandez1010 wrote:

hi, I'm working with RNA-seq data. I reverse complemented sequences in a fastq file using fastx reverse complement function adding the -Q 33 option. Without this option I got this error: "fastx_reverse_complement: Invalid quality score value (char '.' ord 46 quality value -18) on line 8". Now when I align using bowtie (bowtie ../../../E_coli -S -q -p 7 --ff -1 1AR002_noribo_1.fq -2 1AR002_noribo_rv_2.fq >prueba_R1_R2/ff.sam) I get the following error :

Saw ASCII character -54 but expected 33-based Phred qual. terminate called after throwing an instance of 'int'

Anybody has an idea of what could I be making wrong? any suggestion will be appreciated.

bowtie rna-seq fastx • 1.5k views
ADD COMMENTlink written 2.1 years ago by gufernandez1010

Let's start at the beginning, why did you reverse complement the fastq file? Try using the original file instead.

ADD REPLYlink written 2.1 years ago by Devon Ryan85k

Hi, thanks for reply. I should reverse complement the fastq file because i tried to get RPKM using artemis from a paired-end alignment, but artemis don't handle the representation very well of pair-end alignment, showing something like a mirror as a representation of that. So, one option that i have used to get a well representation and metrics before was get reverse complement from R2(pair end file) and then align using --ff option in bowtie, getting a correct representation in artemis. After that getting the corresponding RPKM.

ADD REPLYlink modified 2.1 years ago • written 2.1 years ago by gufernandez1010

So don't use artemis. Give bowtie2 your original unmodified files for alignment and don't use the --ff option anymore.

ADD REPLYlink written 2.1 years ago by Devon Ryan85k

artemis perform easily the RPKM, anyway i follow your advice and perform the count with featureCounts and then estimate the RPKM manually. thanks for reply again.

ADD REPLYlink written 2.1 years ago by gufernandez1010

My bet is that your sequences are based on a old Phred scoring system, e.g. they may be Phred+64 (https://en.wikipedia.org/wiki/FASTQ_format#Encoding).

ADD REPLYlink written 2.1 years ago by Giovanni M Dall'Olio26k

maybe yes, but the only additional option described in fastx to solve problems with encoded is -Q 33 and if i skip this option get the follow :fastx_reverse_complement: Invalid quality score value (char '.' ord 46 quality value -18) on line 8", so i don't know exactly what its the source of trouble.

ADD REPLYlink written 2.1 years ago by gufernandez1010
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