importing paired FASTQ file into qiime had errors. any help?

User Support
21

I am not sure what you mean

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22
  1. how to know which slash I am missing?
  2. regarding the paths u asked me to add in manifest file. which path shall i write? the path in the mini folder I made for training which has six sample (12 file) or its main path in the FASTQ file I downloaded from basespace illumina? I just copied 6 sample to make sure I master the steps
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23

  1. I know you are missing a slash most likely "BACK SLASH \ " because when you run your command, it ran every single line separately.

  2. The path should be exactly where your file is. The path is only relevant in the manifest where the files are sitting currently.

I would just try to get the program to recognize where your manifest is, right now you are not able to run the qiime2 command without having problems, so let’s deal with one problem at a time. Ben

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24

thanks. do I need to make a new directory with this manifest folder in? or work on home directory? maybe that is why it never reads. also, I already added slash at end of every command line to tell the program to run them together

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25

This line tells qiime2 to look in a folder called "import" that is sitting on your desktop. If your manifest file is not in that folder with your samples then it will not work. Ben

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26

no all manifest, metadata, and sample sequences in folder in desktop. I was just thinking why itnever reads them so thought about changing directory to this folder I made

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27

Please be specific, are the fastq.gz samples in that folder called “import” on your desktop? is the manifest file in the same folder?

Can you show me where the files are? Ben

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28

thanks. I am attaching all pics.

  1. I have folder named manifest on desktop (I changed the name and path from import to manifest not to get confused with bad trials)
  2. second picture: content of this folder: manifest file txt format, metadata yml, 12 FASTQ files
  3. Third pic: txt manifest file both R and F in same row and have their new path in manifest folder%20manifest%20file %20desktop %20manifest%20folder%20content
29

qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path /Users/hebahussein/Desktop/manifest --output-path /Users/hebahussein/Desktop/paired-end-demux.qza --input-format PairedEndFastqManifestPhred64V2

run this instead! ben

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30

hebahussein$ qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path /Users/hebahussein/Desktop/manifest --output-path /Users/hebahussein/Desktop/paired-end-demux.qza --input-format PairedEndFastqManifestPhred64V2

Error: Detected invalid character in: ‘SampleData[PairedEndSequencesWithQuality]’

Verify the correct quotes or dashes (ASCII) are being used.

(qiime2-2019.7) a-PC:~ hebahussein$

31

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path /Users/hebahussein/Desktop/manifest --output-path /Users/hebahussein/Desktop/paired-end-demux.qza --input-format PairedEndFastqManifestPhred64V2

copy this instead - there's a weird character qiime2 doesn't like

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32

same . maybe I try to remove the spaces before the double dashes - -?

(qiime2-2019.7) a-PC:~ hebahussein$ qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path /Users/hebahussein/Desktop/manifest --output-path /Users/hebahussein/Desktop/paired-end-demux.qza --input-format PairedEndFastqManifestPhred64V2

Error: Detected invalid character in: ‘SampleData[PairedEndSequencesWithQuality]’

Verify the correct quotes or dashes (ASCII) are being used.

(qiime2-2019.7) a-PC:~ hebahussein$

33

I ran it in terminal, I'm sure it'll work now it was the quotation marks.

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34

but at least we dont get the folder path error anymore.

35

which qoutation so I remove? as it still did not run qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path /Users/hebahussein/Desktop/manifest --output-path /Users/hebahussein/Desktop/paired-end-demux.qza --input-format PairedEndFastqManifestPhred64V2

Error: Detected invalid character in: ‘SampleData[PairedEndSequencesWithQuality]’

Verify the correct quotes or dashes (ASCII) are being used.

36

I think it is because the small ' ' marks are being changed to non-ascii marks.

copy the command in this text file.

copy this into terminal.txt (227 Bytes)

And run it.

To avoid this, you can copy the command into a text file that is in "plain text" and then copy it into terminal. OR you can write the whole command in terminal to avoid this. Ben

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37

I can see you still have single qoutation ’ '. no change but I tried both with and without and still
qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path /Users/hebahussein/Desktop/manifest --output-path /Users/hebahussein/Desktop/paired-end-demux.qza --input-format PairedEndFastqManifestPhred64V2

There was a problem importing /Users/hebahussein/Desktop/manifest:

/Users/hebahussein/Desktop/manifest is not a file.

38

Ok, change this line in that text file from:

/Users/hebahussein/Desktop/manifest

to:

/Users/hebahussein/Desktop/manifest/6manifest.txt

And re-run the entire command with this new line. Ben

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39

so I think the problem in the metadata now
or easier; I change the names of columns in the manifest

There was a problem importing /Users/hebahussein/Desktop/manifest/6manifest.txt:

/Users/hebahussein/Desktop/manifest/6manifest.txt is not a(n) PairedEndFastqManifestPhred64V2 file:

’forward-absolute-filepath’ is not a column in the metadata. Available columns: ‘Forward’, 'Reverse’

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40

rename the column

Forward to forward-absolute-filepath
Reverse to reverse-absolute-filepath

Then rerun! Ben

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