# Stacks2: process\_Radtags won't find paired-end data **URL:** \ **Category:** usegalaxy.eu support\ **Created:** [February 23, 2021, 8:46pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438 "2021-02-23T20:46:27Z")\ **Posts on this page:** 17\ **Page:** 1
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [February 23, 2021, 8:46pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/1 "2021-02-23T20:46:27Z")
Hi everyone! I’m trying to use the tool Stacks2 : process\_radtags to demultiplex paired-end data. I’ve got 2 huge files, R1 and R2, each one at about 45 Gb, in fastqsanger.gz format. The problem I encounter is that when I select “paired-end files” in the first line of the tool, it can’t find my data files. On the other end, if I leave it at “Single-end files”, it finds both of the files with no problem. Should I interlace my two files for this tool to work? Can anyone help me with this small issue? Thanks! ---
**Author:** ![gallardoalba](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/gallardoalba/32/1903_2.png) [@gallardoalba](https://help.galaxyproject.org/u/gallardoalba)\ **Post date:** [February 24, 2021, 10:38am UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/2 "2021-02-24T10:38:56Z")
Hi @Joelle99, you need to create a paired collection. In order to do it you need to select _Operation on multiple datasets_, then select your paired files and finally _Build a Dataset Pair_. ![Screenshot from 2021-02-24 11-36-57](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/6/616bc176aa68c5f972a9af565167c60543d6d98b.png) Regards ---
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [February 24, 2021, 2:16pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/3 "2021-02-24T14:16:39Z")
Hi, and thank you for your answer! Sadly, I had already tried beforehand to build a dataset pair, but it still can’t find my file with my two reads… ---
**Author:** ![innovate-invent](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/innovate-invent/32/734_2.png) [@innovate-invent](https://help.galaxyproject.org/u/innovate-invent)\ **Post date:** [February 24, 2021, 5:30pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/4 "2021-02-24T17:30:39Z")
Have you double checked that the format that was assigned by Galaxy is what the tool requires? ---
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [February 24, 2021, 6:29pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/5 "2021-02-24T18:29:34Z")
Yes I did ! Here’s what they ask for : ![image](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/d/de033a5da66bc752e9cc05708783132096364842.png) Both of my files are in fastqsanger.gz, which should then be fine I guess… ---
**Author:** ![innovate-invent](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/innovate-invent/32/734_2.png) [@innovate-invent](https://help.galaxyproject.org/u/innovate-invent)\ **Post date:** [February 24, 2021, 6:49pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/6 "2021-02-24T18:49:15Z")
Is that the value Galaxy assigned to the uploaded files? You can double check by clicking the edit attributes pencil icon on the dataset, then going to the “Datatypes” tab ---
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [February 24, 2021, 6:55pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/7 "2021-02-24T18:55:38Z")
Yes it is! Here’s one file for example : ![image](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/d/d9b0dd71d45bf4256f17c5a99c4f0889f9579051.png) ---
**Author:** ![innovate-invent](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/innovate-invent/32/734_2.png) [@innovate-invent](https://help.galaxyproject.org/u/innovate-invent)\ **Post date:** [February 24, 2021, 7:08pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/8 "2021-02-24T19:08:53Z")
Can you do a screen cap of the collection you had created? ---
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [February 24, 2021, 7:15pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/9 "2021-02-24T19:15:03Z")
There you go! ![image](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/8/85fbbc2c90406f23cc45e8a31db289b0e926f71c.png) ---
**Author:** ![innovate-invent](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/innovate-invent/32/734_2.png) [@innovate-invent](https://help.galaxyproject.org/u/innovate-invent)\ **Post date:** [February 24, 2021, 7:29pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/10 "2021-02-24T19:29:43Z")
I’ll admit I have zero experience working with paired collections. Are you sure this is a ‘list:pair’ and not somehow a ‘pair’. Someone else is going to have to weigh in on this. ---
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [February 24, 2021, 7:35pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/11 "2021-02-24T19:35:50Z")
Well, I followed @gallardoalba instructions so I guess it must be okay? To be honest, this is my first time analysing sequencing data, so I’m just starting to learn how Galaxy (and Rad-Seq analysis) works… ---
**Author:** ![yvanlebras](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/yvanlebras/32/1813_2.png) [@yvanlebras](https://help.galaxyproject.org/u/yvanlebras)\ **Post date:** [March 1, 2021, 10:31am UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/12 "2021-03-01T10:31:48Z")
Hi Joelle! Thank you for reporting your issue. It seems to me there is something wrong somewhere… First, on the [process radtags tool](https://ecology.usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/iuc/stacks_procrad/stacks_procrad/1.46.0) (-\> maybe it can be of interest for us to know if are you using the last version of the tool ? / in which Galaxy instance ?) , if you have paired-end data, you need to select as input, R1 file(s) (or collection) in the first “paired-end reads infile(s)” ““box”” and R2 file(s) (or collection) in the second “paired-end reads infile(s)” ““box””. So here, it seems to me you created a collection with both R1 and R2 files, so it’s a good dataset pair ;), but with this tool, you need to use separately R1 and R2. After saying that, normaly this tools is not “looking” at the name or content of the files when you are “just” selecting it… so… normally, if the datasets formats are ok, the tool will display these datasets on the “select box”… One exaplanation for ma can be that there is an issue using fastqsanger.gz WHEN in a collection… so MAYBE, you need to use Fastqsanger or fastqsanger.gz input files (so not in a collection) OR use fastqsanger files if in a collection… I have to test it! So, please, can you try to “just” select your fastqsanger.gz from your history (not in collection) ? And please, pay attention to the fact that your files need to be named something like"name\_R1\_001.fastq" and “name\_R2\_001.fastq” so the execution of the tool can work 😉 Additionally, don’t hesitate to share your history so I can have a look. ---
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [March 1, 2021, 2:06pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/13 "2021-03-01T14:06:03Z")
Hi! Thanks a lot for your return. So I first used Stacks:process\_radtags (Version 1) with no problem with the same files. But, for any reason, it won’t detect my files (even if the files are separate, i.e not in a collection) with Stacks **2** :process\_radtags (Version 2). I am working in the EU instance of Galaxy. Here are my two files : ![image](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/0/08e2e8178f118fb9231169a1675dc959b0254ba0.png) Thank you! 🙂 ---
**Author:** ![yvanlebras](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/yvanlebras/32/1813_2.png) [@yvanlebras](https://help.galaxyproject.org/u/yvanlebras)\ **Post date:** [March 1, 2021, 3:09pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/14 "2021-03-01T15:09:16Z")
OK! It appears there is a pb with the last [stacks2 process radtags tool](https://ecology.usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/iuc/stacks2_procrad/stacks2_procrad/2.4+galaxy1) … And I don’t know why, there is no more process radtags tool named as the [last one I used](https://ecology.usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/iuc/stacks_procrad/stacks_procrad/1.46.0). Can you try to access and use this last version who is working for me on eu but seems to be ~hidden : the [last procrad tool who is working properly for me](https://ecology.usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/iuc/stacks_procrad/stacks_procrad/1.46.0) ---
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [March 1, 2021, 3:30pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/15 "2021-03-01T15:30:16Z")
Hi again 🙂 Yes, this is also the version I used (1.46), it works well! I based my analysis on this one since I wasn’t able to make the new one (version 2.4) work. I just wished to be able to use the new one in the future as it seems a few improvements were done. But if there’s a true bug, I at least know that it’s not my fault if I can’t get it to work! I’ll keep checking in the future to see if the bug is resolved and the tool usable for my analysis. Thank you! 🙂 ---
**Author:** ![yvanlebras](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/yvanlebras/32/1813_2.png) [@yvanlebras](https://help.galaxyproject.org/u/yvanlebras)\ **Post date:** [March 1, 2021, 6:08pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/16 "2021-03-01T18:08:13Z")
Hi again 😉 In fact, it appears that to work properly with the 2.4 tool version, you need to use the “Build list of Dataset Pairs” function, not “Build a Dataset Pair”, even if you only have one pair… I think this will be ok! ---
**Author:** ![Joelle99](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/joelle99/32/2173_2.png) [@Joelle99](https://help.galaxyproject.org/u/Joelle99)\ **Post date:** [March 1, 2021, 6:54pm UTC](https://help.galaxyproject.org/t/stacks2-process-radtags-wont-find-paired-end-data/5438/17 "2021-03-01T18:54:40Z")
It does work!!! Thank you very much for your time and answer! I’ll be able to test this new version now 🙂 Have a good day/evening!