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

Hi. I activated qiime 2 2019, prepared a folder named '6 sample file' to try data import which has metadata, manifest, and 6 data sequences as in the picture. I received this error. maybe as manifest and metadata are not qza (I read the tutorial but could not find how to convert into qza. Thanks a lot
6%20sample%20file There was a problem with the command:
(1/1?) no such option: -q
(qiime2-2019.7) a-PC:~ hebahussein$
(qiime2-2019.7) a-PC:~ hebahussein$ qiime tools import \

--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path 6 sample import
--output-path paired-end-demux.qza
--input-format PairedEndFastqManifestPhred64V2

input file’s name should be complete i.e. 6 sample manifest.csv. If this doesn’t work, then please try converting .csv into .txt. I am not sure if .csv is an accepted file, hence, incase if problem still persists after using the complete name then you need to convert the format and see what happens.

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I see that t you are working in the virtual environment? Could you please try using this command and see what happens:
qiime tools import
–type ‘SampleData[PairedEndSequencesWithQuality]’
–input-path /6 sample file/6 sample manifest.csv
–output-path /6 sample file/paired-end-demux.qza
–input-format PairedEndFastqManifestPhred64V2

The above should help. If this doesn’t work then please convert the .csv file into .txt and repeat the above commands by using the .txt file name.

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I read the tutorial many times but still get errors. Do I need to make a folder with metadata file and manifest file only to import to qiime? Do I need to include in the folder the individual FASTQ files (2 per each sample) as attached in the picture? What are the required format of all the files? I have metadata in yml, manifest in excel or CSV, and individual sequence files in qz extension. Anyone has the exact codes to import these files for quality analysis and denosing? I am struggling with this step for too long and tried many extensions so will appreciate your help. 73021842_1718016368328907_2880760596947533824_n

Screen shot what is in your manifest file please

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Thanks ben attached my manifest. three columns. excel or cvs extensionmanifest%20

Hi @HebaHussein-1981,

I merged these two topics because they seem to be addressing the same problem. This is a gentle reminder to try and keep topics together; it makes it easier for everyone to use the forum and keeps things consistent.

Best,
Justine

3 Likes

manifest.txt (866 Bytes)

I made the manifest for you.

You have to do a little work, you have to replace the filepath where I wrote "/NEEDS/FULL/FILEPATH/" Ben

edit: the forward and reverse file has to be in the same row - sampleID i am assuming Rahe001-UC etc. I am assuming this is an ulcerative colitis study?

The command in Qiime2 should direct to this manifest.txt file - the manifest.txt will tell QIIME2 where to find the files.

To answer your questions:

  1. They are 12 individidual files - they just happen to be forward and reverse for 1 sample
  2. You should use what format you like, I use TXT tab delimited as it is what I was trained in and what i know.
  3. Again, I should reemphasize, these are still 12 files, but QIIME2 needs to know that they belong to 6 samples.
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Thanks so I need to write the full path for each individual file? also, to import to qiime, shall I import this manifest file as txt and metadata as yml? no need for the 12 individual files for 6 samples (6 forward and 6 reverse)?

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no not ulcerative colitis. I dont know how to make the forward and reverse in same row and when I write the path, shall i write it wish slashes as you did?

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Yes, remember I showed you a trick to find the full path name, if not, it’s right clicking on a file in a MAC then HOLDING alt/option you have an option of selecting the full filepath. Ben

2 Likes

I dont remember the trick. I click on file, get info, where, copy and paste in word to know the path

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On a mac, you can also take a file/folder in finder, drag the icon to the terminal, and get the full path.

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source

edit: No love for hacking the gibson :frowning:

2 Likes

Thanks ben. I changed the extension to txt. I made the reverse and forward in one line. I added the path. I pasted this code in qiime environment and got this error. I am attaching the screenshots unzip -q pe-64.zip

qiime tools import **

--type 'SampleData[PairedEndSequencesWithQuality]' **

--input-path /Users/hebahussein/Desktop/6 sample import **

--output-path paired-end-demux.qza **

--input-format PairedEndFastqManifestPhred64V2

the error I got:
There were some problems with the command:
(1/2) Invalid value for "--input-path": Path "/Users/hebahussein/Desktop/6"
does not exist.
(2/2) Got unexpected extra arguments (sample import)
(qiime2-2019.7) a-PC:~ hebahussein$

new%20import 6 s manifest w path.txt (1.1 KB)

1 Like

thanks. I changed the extension to txt. I made the reverse and forward in one line. I added the path. I pasted this code in qiime environment and got this error. I am attaching the screenshots unzip -q pe-64.zip

qiime tools import **

--type 'SampleData[PairedEndSequencesWithQuality]' **

--input-path /Users/hebahussein/Desktop/6 sample import **

--output-path paired-end-demux.qza **

--input-format PairedEndFastqManifestPhred64V2

the error I got:
There were some problems with the command:
(1/2) Invalid value for "--input-path": Path "/Users/hebahussein/Desktop/6"
does not exist.
(2/2) Got unexpected extra arguments (sample import)
(qiime2-2019.7) a-PC:~ hebahussein$

new%20import 6 s manifest w path.txt (1.1 KB)

2 Likes

Rename your file, as a rule, never have a file name with spaces.

Change your file to 6_s_manifest_w_path.txt instead of “6 s manifest w path.txt”. Ben

1 Like

thanks Yes I did remove all spaces from the manifest file and even changed the folder into one word so no need for dashes or spaces and got same errors. Do I need to mkdir new folder and and change directory to it and put: metadata: 12 file sequence: and manifest? I even tried and did not work. There were some problems with the command:

(1/4) Missing option “–type”.

(2/4) Missing option “–input-path”.

(3/4) Missing option “–output-path”.

(4/4) Got unexpected extra argument ( )

(qiime2-2019.7) a-PC:~ hebahussein$ --type ‘SampleData[PairedEndSequencesWithQuality]’ \

-bash: --type: command not found

(qiime2-2019.7) a-PC:~ hebahussein$ --input-path /Users/hebahussein/Desktop/import \

-bash: --input-path: command not found

(qiime2-2019.7) a-PC:~ hebahussein$ --output-path /Users/hebahussein/Desktop/paired-end-demux.qza \

-bash: --output-path: command not found

(qiime2-2019.7) a-PC:~ hebahussein$ --input-format PairedEndFastqManifestPhred64V2

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hm, I think you’re missing a slash someplace.

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

the manifest file is within the “import” folder correct?

1 Like

yes how to know which slash? also, I will remake the manifest file. maybe when we changed the name of the manifest file and folder and removed the spaces, it did not recognize them