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Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
08-Apr-2015, 04:35 PM
Post: #1
Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
If you encounter an error like "Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual" in an error report check if --galaxy_input_format="fastqillumina" in the job command line. This error can appear when processing a dataset using the "Remove rRNA using Bowtie" tool. The easiest option is to modify the file format (i.e. datatype) using the edit attributes (pencil icon) in your history display for the FASTQ file. The format most likely should be fastqsanger. You should also ensure you specify the correct format when you upload the file.
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07-Nov-2016, 11:41 AM
Post: #2
RE: Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
I received this error...

Error aligning sequence. Saw ASCII character 51 but expected 64-based Phred qual. Try not specifying --solexa1.3-quals/--phred64-quals. Command: bowtie -q -p 1 -S -v 3 -n 2 -e 70 -l 25 --norc --maxbts 125 -k 1 --un /mnt/workspace/DATA/galaxy/galaxy-dist/

I tried changing the file format to fastqsanger, but then the file disappeared from the dropdown list of FASTQ files that could be seleceted. So, I changed it back to fastqillumina and the file came back to the list. But still received the error when executing the rRNA removal. Please forgive my incompetence, I'm very new to this and trying to follow the protocol using "RiboGalaxy_published_slides.pdf"

Any help would be much appreciated.
Thank you
Ben
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06-Dec-2016, 09:08 PM
Post: #3
RE: Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
Dear Ben,
let me know if you got this issue resolved so that I can post the solution here for the benefit of other users ?

Regards
jamesp
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19-Jan-2018, 04:36 AM (This post was last modified: 19-Jan-2018 04:36 AM by 06dalton.)
Post: #4
RE: Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
(06-Dec-2016 09:08 PM)jamespa Wrote:  Dear Ben,
let me know if you got this issue resolved so that I can post the solution here for the benefit of other users ?

Regards
jamesp

Dear Jamespa, I also encountered the same error. As instructed from the website, "Bowtie accepts files in Sanger FASTQ format. Use the FASTQ Groomer to prepare your files." Thus, I first used the FASTQ Groomer (from Galaxy website toolkit) to convert my files into FASTQ, and then I changed the files attribute into "fastqsanger". Now, I can use Bowtie to remove the rRNA successfully.

Hope this will help.
Best,
06dalton
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18-Feb-2018, 10:35 AM
Post: #5
RE: Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
Having the same issue here, or at least it looks like it. But this did nothing for me. The problem is still there. Any other suggestions?
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20-Feb-2018, 10:31 AM
Post: #6
RE: Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
Hi,

If changing the file format to fastqsanger (by clicking on 'edit attributes' and then selecting fastqsanger under the 'datatypes' tab), does not resolve the issue, can you send your error to the RiboGalaxy team from within RiboGalaxy so that we have a trace of the history? When we receive your email, we will then try to troubleshoot the issue to determine what is causing the error.

Kind regards,
Audrey
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18-Apr-2018, 11:40 AM
Post: #7
RE: Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
Hi,
I'm not sure if this is the right thread to post my question, I've been using ribogalaxy for some time now and I think it's an awesome tool, thank you very much.

So my question is about bowtie alignments. I need to use an aligner sensitive to splicing events and I've read that bowtie is not, and that's why people use Tophat after bowtie. Does the ribogalaxy platform have the Tophat incorporated as well, or is it just the bowtie? I've also read somewhere that bowtie works better (splice-wise) when you align to transcriptome rather than genome. Is that true?

Thanks for any replies,

all the best,

Katerina
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19-Apr-2018, 08:21 AM
Post: #8
RE: Error aligning sequence. Saw ASCII character 35 but expected 64-based Phred qual
Hi Katerina,

Thanks for your interest in RiboGalaxy.

You are correct in that bowtie is not splice aware. Currently it is the only short read alignment tool that we provide in RiboGalaxy. We have found bowtie to be among the most accurate when mapping Ribo-seq reads to the transcriptome. When mapping to a genome, bowtie will not map reads that span splice junctions. However, mapping short Ribo-seq reads (~30nt) across splice junctions is difficult anyhow.

The public Galaxy platform (https://usegalaxy.org/) has a number of splice aware alignment tools including TopHat which may be of interest to you.

All the best,
Audrey
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