bowtie "Saw ASCII character -54 but expected 33-based Phred qual." after -Q 33 fastx reverse complement
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4.6 years ago

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.

RNA-Seq bowtie fastx • 2.9k views
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Let's start at the beginning, why did you reverse complement the fastq file? Try using the original file instead.

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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.

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

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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.

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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).

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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.

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