Choosing the correct phred score when importing data

Hello @colinbrislawn ,

I have obtained a set of fastq files from an ion torrent sequencing machine. I came across an application note on ion torrent which said that semiconductor sequencing produces a raw accuracy rate of over 99% which is Q20. Due to this reason, I decided to input my data in SingleEndFastqManifestPhred33V2 format.
The command I used is given below:

qiime tools import **
--type 'SampleData[SequencesWithQuality]' **
--input-path /home/Brigitta/manifestfile1.txt **
--output-path single-end-demux.qza **
--input-format SingleEndFastqManifestPhred33V2

However, my single-end-demux.qzv file shows the danger message, " Danger: Some of the forward PHRED quality values are out of range. This is likely because an incorrect PHRED offset was chosen on import of your raw data. You can learn how to choose your PHRED offset during import in the importing tutorial."

I tried importing the data in SingleEndFastqManifestPhred64V2 format but I didn't receive an output file. Instead, it said that the Phred score was out of range (range: 0-62).

Can I please get a solution to this?

Thank you in advance,

Hello @Brigitta1,
This question is pretty similar to the other question you have open. In the future if you could wait for a moderators response and then ask the related question that would be appreciated. It would help us keep the forum tidy!

I am not super familiar with Ion Torrent sequencing but it looks like S5 runs include quality scores that could be above 40, which would explain the error you are getting when you run SingleEndFastqManifestPhred33V2 format.

I found this link and it might be helpful: phred 33 from Ion S5?

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I thought opening two questions would keep the forum tidy because one of my questions was on importing data and the other was about using DADA2. Sorry about that @cherman2

Thank you so much for this piece of information. Are you suggesting that I set my Phred score to 40 or >40 when importing data?

My bad! I thought your posts were more related than they are.

I would look into running the commands in the link I sent. They used a command that seemed to reduce their quality scores to < 40 that way you can import as a SingleEndFastqManifestPhred33V2 format.

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No worries!

Thank you. I went through the link you sent. They have suggested a different trimming tool that can be used. However, I would like to reduce the quality scores to <40 and import it as a SingleEndFastqManifestPhred33V2 format because I would like to use QIIME2 to trim my sequences instead of using a different trimming tool. Could you please suggest a command or a tool that I could use?

Hi @Brigitta1,
I talked to another person in my lab and he suggested that although this looks kinda funky and had that warning, these sequences are about as good as you can get. So no addition steps are needed. I would just trim carefully but you should be good!

Hope that helps!


Thank you so much @cherman2

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