# Issue importing Paired end sequences with quality
**URL:** https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256
**Category:** Technical Support
**Tags:** import, fastq-manifest
**Created:** [November 4, 2021, 5:49pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256 "2021-11-04T17:49:03Z")
**Posts on this page:** 12
**Page:** 1
### Author:  [@lautaro.rostoll](https://forum.qiime2.org/u/lautaro.rostoll)
#### Post date: [November 4, 2021, 5:49pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/1 "2021-11-04T17:49:03Z")
Hello Everyone,
I am trying to import Paired end sequences with quality using a manifest (attached here an example with just a few files. [Manifest.txt](https://forum.qiime2.org/uploads/short-url/tFxARfcbDwKUtP0tCb3h1n6bRSe.txt) (547 Bytes)
I am using putty to access the server from my university and run qiime with conda.
The files are in a folder within the server and the path to the folder where the fastq files are is: home/lrostoll/qiime2/neo
When I try to import the files with this code:
qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path Manifest.txt
--output-path demux-paired-end.qza
--input-format PairedEndFastqManifestPhred33V2
I get this error: Manifest.txt is not a(n) PairedEndFastqManifestPhred33V2 file:
Filepath on line 1 and column "forward-absolute-filepath" could not be found ($home/lrostoll/qiime2/neo/155\_S15\_L001\_R1\_001.fastq.gz) for sample "1-17-IL-C".
I would really appreciate some help with this
thanks,
Lautaro
---
### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [November 5, 2021, 2:20pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/2 "2021-11-05T14:20:17Z")
Hello Lautaro,
I took a look at your manifest file, and it looks good, except for the line endings.
(Windows and Linux use different ways to note a
new
line
of
text.)
Once you convert to the Linux format by saving it with a text editor or using a command like
`dos2unix Manifest.txt`
then this should work great!
You are not the first to have this issue! More info here:
> [@FASTQ Files Importing Error](https://forum.qiime2.org/t/fastq-files-importing-error/6412/19):
>
> If you use Excel to make your CSV, it will insert CRLF line breaks (whether or not you are using windows, I believe). So you need to either just open in textedit and use find/replace to fix the line breaks, or use mac2unix or a program like that to convert the carriage returns. So Excel is okay — just make sure to convert the line endings. This will be an issue with making sample metadata files, too, not only manifest files.
---
### Author:  [@lautaro.rostoll](https://forum.qiime2.org/u/lautaro.rostoll)
#### Post date: [November 5, 2021, 7:44pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/5 "2021-11-05T19:44:19Z")
> [@colinbrislawn](#):
>
> dos2unix Manifest.txt
Hi Colin,
thanks so much for your response. I tried converting the file with the dos2unix command and it was converted successfully. However, I still get the same error message.
[Manifest.txt](https://forum.qiime2.org/uploads/short-url/eo4w0RA8XI7Sr7S3vBIbMq87N2S.txt) (542 Bytes)
Here is the new manifest file.
Is there anything else that could be happening?
thanks,
Lautaro
---
### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [November 5, 2021, 8:40pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/6 "2021-11-05T20:40:16Z")
Hello Lautaro,
I have made a mistake! I missed something important in your original message:
> [@lautaro.rostoll](#):
>
> Filepath on line 1 and column "forward-absolute-filepath" could not be found ($home/lrostoll/qiime2/neo/155\_S15\_L001\_R1\_001.fastq.gz) for sample "1-17-IL-C".
This shows an issue with finding the file, not with line endings.
Double-check that file path and see if you can get it pointing to the right file. (For example, on my computer, `$HOME` points to my home directory, but `$home` is not set at all!)
Let us know how it goes,
Colin
---
### Author:  [@lautaro.rostoll](https://forum.qiime2.org/u/lautaro.rostoll)
#### Post date: [November 6, 2021, 5:35pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/7 "2021-11-06T17:35:48Z")
Hi Colin,
I tried changing $home/ to $HOME/ and it didn't work either.
Here is my file path: /home/lrostoll/qiime2/neo
I tried doing HOME/lrostoll/qiime2/neo, and /HOME/lrostoll/qiime2/neo, and also the same with home in lower case. non of them seems to work. 🤷♂️
Here is the manifest again:
[Manifest.txt](https://forum.qiime2.org/uploads/short-url/nthxbwecttzK4bz9YpYkLrFqTz1.txt) (547 Bytes)
And here is the error I am getting:
There was a problem importing Manifest.txt:
Manifest.txt is not a(n) PairedEndFastqManifestPhred64V2 file:
Filepath on line 1 and column "forward-absolute-filepath" could not be found ($HOME/lrostoll/qiime2/neo/155\_S15\_L001\_R1\_001.fastq.gz) for sample "1-17-IL-C".
Any ideas of what I am doing wrong?
thanks!
Lautaro
---
### Author:  [@lautaro.rostoll](https://forum.qiime2.org/u/lautaro.rostoll)
#### Post date: [November 6, 2021, 5:59pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/9 "2021-11-06T17:59:00Z")
I also tried changing to Phred64V2 and also didn't work.
I also made sure that the format was UTF-8 without BOM and used a text editor to double check that.
I have no idea why it isn't working 😩
---
### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [November 7, 2021, 3:54pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/11 "2021-11-07T15:54:26Z")
> [@lautaro.rostoll](#):
>
> I have no idea why it isn't working 😩
Well, the good news is that we are still dealing with the same error. (So nothing spooky is going on 👻 )
> [@lautaro.rostoll](#):
>
> Filepath on line 1 and column "forward-absolute-filepath" could not be found
Let's take a look in these folders and see what's inside of them. Run each of these commands and post the full output:
```bash
cd $HOME; pwd
ls -lah $HOME
ls -lah $HOME/qiime2/neo/
ls -lah $HOME/lrostoll/qiime2/neo/
```
---
### Author:  [@lautaro.rostoll](https://forum.qiime2.org/u/lautaro.rostoll)
#### Post date: [November 7, 2021, 4:46pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/12 "2021-11-07T16:46:34Z")
Here it is:
> [@colinbrislawn](#):
>
> `ls -lah $HOME`
```auto
(qiime2-2021.2) lrostoll@signal[11:43][~]$ ls -lah $HOME
total 91M
drwx--x--x 19 lrostoll lrostoll 4.0K Mar 15 2021 .
drwxr-xr-x 528 root root 16K Nov 2 11:34 ..
-rw-r--r-- 1 lrostoll lrostoll 18 Oct 30 2018 .bash_logout
-rw-r--r-- 1 lrostoll lrostoll 193 Oct 30 2018 .bash_profile
-rw-r--r-- 1 lrostoll lrostoll 919 Jan 27 2021 .bashrc
-rw-r----- 1 lrostoll lrostoll 0 Oct 25 2019 build
drwxrwxr-x 5 lrostoll lrostoll 63 Jan 27 2021 .cache
-rw-r----- 1 lrostoll lrostoll 19K Oct 5 2019 comp1.cpp
-rw-r----- 1 lrostoll lrostoll 402 Oct 5 2019 comp1.csi
-rw-r----- 1 lrostoll lrostoll 419 Oct 5 2019 comp1.csl
-rw-r----- 1 lrostoll lrostoll 1016 Oct 5 2019 comp1.rrr
drwxr-x--- 2 lrostoll lrostoll 38 Jan 26 2021 .conda
drwxrwxr-x 5 lrostoll lrostoll 71 Jan 27 2021 .config
-rw-r----- 1 lrostoll lrostoll 301 Oct 25 2019 consoleError.log
-rw-r----- 1 lrostoll lrostoll 424 Oct 25 2019 consoleStatus.log
-rw-r----- 1 lrostoll lrostoll 647 Oct 25 2019 cori1
-rw-r----- 1 lrostoll lrostoll 694 Oct 25 2019 cori1.csi
-rw-r----- 1 lrostoll lrostoll 647 Oct 21 2019 cori1.csl
drwxr-x--- 2 lrostoll lrostoll 83 Jan 27 2021 .electron
-rw-r--r-- 1 lrostoll lrostoll 334 Oct 30 2018 .emacs
drwx------ 3 lrostoll lrostoll 36 Sep 30 2019 .emacs.d
-rw------- 1 lrostoll lrostoll 111K Apr 22 2021 .history_eramosa
-rw------- 1 lrostoll lrostoll 119K Nov 6 17:03 .history_signal.aps.uoguelph.ca
-rw------- 1 lrostoll lrostoll 475 Oct 21 2019 .InterpConsole_history
drwxr-x--- 2 lrostoll lrostoll 32 Feb 22 2021 .keras
drwx------ 3 lrostoll lrostoll 27 Sep 30 2019 .local
-rw-r----- 1 lrostoll lrostoll 65 Oct 21 2019 Makefile
-rw-r----- 1 lrostoll lrostoll 66 Oct 21 2019 Makefile~
drwxr-x--- 16 lrostoll lrostoll 302 Jan 26 2021 miniconda3
-rw-r----- 1 lrostoll lrostoll 90M Nov 23 2020 Miniconda3-latest-Linux-x86_64.sh
drwxr-xr-x 4 lrostoll lrostoll 51 Jul 20 2017 .mozilla
drwxr-x--- 4 lrostoll lrostoll 119 Jan 27 2021 .npm
drwxr-x--- 8 lrostoll lrostoll 4.0K Jan 27 2021 .nvm
drwxr----- 3 lrostoll lrostoll 27 Jan 27 2021 .pki
drwxr-s--- 2 lrostoll apache 32 Sep 26 2019 public_html
drwxr-x--- 5 lrostoll lrostoll 100 Feb 19 2021 q2studio-2020.11.0
drwxr-x--- 12 lrostoll lrostoll 8.0K Nov 4 09:45 qiime2
-rw-r----- 1 lrostoll lrostoll 9.3K Jan 26 2021 qiime2-2020.11-py36-linux-conda.yml
-rw-r----- 1 lrostoll lrostoll 9.4K Mar 15 2021 qiime2-2021.2-py36-linux-conda.yml
drwxr-x--- 4 lrostoll lrostoll 4.0K Jan 28 2021 qiime2-moving-pictures-tutorial
drwx------ 2 lrostoll lrostoll 100 Jan 26 2021 .ssh
-rw-r--r-- 1 lrostoll lrostoll 1.5K Nov 19 2004 .Xdefaults
-rw-r--r-- 1 lrostoll lrostoll 607 Nov 19 2004 .Xresources
```
```auto
**ls -lah $HOME/qiime2/neo/**
(qiime2-2021.2) lrostoll@signal[11:44][~]$ ls -lah $HOME/qiime2/neo/
total 933M
drwxr-x--- 2 lrostoll lrostoll 4.0K Nov 6 13:19 .
drwxr-x--- 12 lrostoll lrostoll 8.0K Nov 4 09:45 ..
-rw-r----- 1 lrostoll lrostoll 80M Nov 1 14:35 155_S15_L001_R1_001.fastq
-rw-r----- 1 lrostoll lrostoll 80M Nov 1 14:35 155_S15_L001_R2_001.fastq
-rw-r----- 1 lrostoll lrostoll 155M Nov 1 14:36 157_S16_L001_R1_001.fastq
-rw-r----- 1 lrostoll lrostoll 155M Nov 1 14:36 157_S16_L001_R2_001.fastq
-rw-r----- 1 lrostoll lrostoll 119M Nov 1 14:35 158_S17_L001_R1_001.fastq
-rw-r----- 1 lrostoll lrostoll 119M Nov 1 14:35 158_S17_L001_R2_001.fastq
-rw-r----- 1 lrostoll lrostoll 115M Nov 1 14:35 159_S18_L001_R1_001.fastq
-rw-r----- 1 lrostoll lrostoll 114M Nov 1 14:35 159_S18_L001_R2_001.fastq
-rw-r----- 1 lrostoll lrostoll 547 Nov 4 13:52 Manifest.csv
-rw-r----- 1 lrostoll lrostoll 542 Nov 6 16:51 Manifest.txt
```
```auto
**ls -lah $HOME/lrostoll/qiime2/neo/**
(qiime2-2021.2) lrostoll@signal[11:45][~]$ ls -lah $HOME/lrostoll/qiime2/neo/
ls: cannot access /home/lrostoll/lrostoll/qiime2/neo/: No such file or directory
```
---
### Author:  [@lautaro.rostoll](https://forum.qiime2.org/u/lautaro.rostoll)
#### Post date: [November 7, 2021, 4:58pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/13 "2021-11-07T16:58:13Z")
I just try again using this path in the manifest: $HOME/qiime2/neo/ and still get the same error:
There was a problem importing Manifest.txt:
Manifest.txt is not a(n) PairedEndFastqManifestPhred33V2 file:
Filepath on line 1 and column "forward-absolute-filepath" could not be found ($HOME/qiime2/neo/155\_S15\_L001\_R1\_001.fastq.gz) for sample "1-17-IL-C".
Here is the manifest:
[Manifest.txt](https://forum.qiime2.org/uploads/short-url/k8LmzmThhukHLDwHBF2XfND1dgp.txt) (470 Bytes)
When I open on a note editor it tells me that the encoding is: 1252 (ANSI - Latin I)
Should it be UTF-8? Can this be the problem?
---
### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [November 7, 2021, 5:01pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/14 "2021-11-07T17:01:20Z")
We are so close!
First, we now have the correct path to the folder: `$HOME/qiime2/neo/`
> [@lautaro.rostoll](#):
>
> could not be found ($HOME/qiime2/neo/155\_S15\_L001\_R1\_001.fastq.gz
Well that makes sense; as we saw above, your files in that folder end with `.fastq` not `.fastq.gz`.
Try taking out the `.gz` from your manifest, and try it one more time!
---
### Author:  [@lautaro.rostoll](https://forum.qiime2.org/u/lautaro.rostoll)
#### Post date: [November 7, 2021, 5:26pm UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/15 "2021-11-07T17:26:09Z")
IT WORKED!!!!
I removed the .gz and it worked.
Thanks so much Colin!
---
### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [December 9, 2021, 1:00am UTC](https://forum.qiime2.org/t/issue-importing-paired-end-sequences-with-quality/21256/17 "2021-12-09T01:00:42Z")
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