# Download and Extract reads in FASTQ format from NCBI SRA in workflow issue **URL:** \ **Category:** usegalaxy.org support\ **Tags:** troubleshooting, transcriptomics, collections, tool-help, collection-shape\ **Created:** [October 27, 2025, 4:12pm UTC](https://help.galaxyproject.org/t/download-and-extract-reads-in-fastq-format-from-ncbi-sra-in-workflow-issue/16430 "2025-10-27T16:12:08Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![erdavenport](https://avatars.discourse-cdn.com/v4/letter/e/df788c/32.png) [@erdavenport](https://help.galaxyproject.org/u/erdavenport)\ **Post date:** [October 27, 2025, 4:12pm UTC](https://help.galaxyproject.org/t/download-and-extract-reads-in-fastq-format-from-ncbi-sra-in-workflow-issue/16430/1 "2025-10-27T16:12:08Z")
Hi! I am teaching a course where we run through an RNA-seq analysis where we take one sample through multiple steps (Download using “Download and Extract reads in FASTQC format from NCBI SRA”, run FastQC, trimmomatic, run FastQC again, HISAT2, featureCounts), extract a workflow, and then apply that workflow to 5 more samples. However, I’m having an issue building a workflow because of how the initial Download step is working. It seems that the Download step is creating two outputs in my history that are lists with single-end data (a list with 1 fastqsanger.gz dataset) and paired-end data (a list with 0 pairs). There’s a hidden file in my history that’s saved as a fastqsanger.gz. If I just leave the lists in my history, those lists aren’t detected as a relevant input file type by FastQC. If I manually un-hide the fastqsanger.gz, that is. I can complete the FastQC and all other steps fine. However, if I extract a workflow and try to run on additional samples, those invocations break, because the workflow can’t automatically go from the output of “Download and Extract reads in FASTQC format from NCBI SRA” to FastQC. ![Screenshot 2025-10-27 at 11.56.16 AM](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/6/6d4da3fa136a2b6c029dda8cc8b8d97df9ead440.png) I’m probably missing something really obvious, but how can I get these steps to play nicely together in a workflow? Is there something I should be doing to those lists after data is downloaded to get them as detectable automatically by FastQC? Here’s my current workflow, showing that Downloading, FastQC, and the rest of the workflow are unconnected. If I try to manually connect them, I get an error box that pops up saying “Can’t map over this input with output collection type” ![Screenshot 2025-10-27 at 12.08.50 PM](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/d/d16b444858fecfffac3442080a056147f84897a8.png) I ran through this in class two years ago and didn’t have this issue. It used to be that the output of the “Download and Extract reads in FastQC format from NCBI SRA” was the fastq file itself, so it seems that this is something that has changed about how the output of this tool is saved. ![Screenshot 2025-10-27 at 12.02.59 PM](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/e/eb04a5cd5532b05cd0ce22d37019fce98268cbaf.png) I’m getting the lists using the current version of “Download…” (Galaxy Version 3.1.1+galaxy1), with the last time I taught the course using (Galaxy Version 2.10.8+galaxy0). Any guidance you could offer would be greatly appreciated! Thanks, Emily ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [October 27, 2025, 5:33pm UTC](https://help.galaxyproject.org/t/download-and-extract-reads-in-fastq-format-from-ncbi-sra-in-workflow-issue/16430/2 "2025-10-27T17:33:37Z")
Welcome @erdavenport Thanks for sharing all these details, and I think we’ll be able to help! There are two similar tools for NCBI SRA fastq data download. The underlying scripts are based on the same NCBI’s toolkit but the options are configured to work _slightly_ differently. I’m going to define the tools first, then address your question. Feel free to jump to the bottom then use the extra help as a reference instead. 🙂 ### Tool Help What it does? This tool extracts data (in [fastq](https://en.wikipedia.org/wiki/FASTQ_format) format) from the Short Read Archive (SRA) at the National Center for Biotechnology Information (NCBI). It is based on the [fasterq-dump](https://github.com/ncbi/sra-tools/wiki/HowTo:-fasterq-dump) utility of the SRA Toolkit. The following applies: > - if data is paired-ended (or mate-pair) the tool will generate a collection of file pairs, in which each element will be a pair of [fastq](https://en.wikipedia.org/wiki/FASTQ_format) files containing forward and reverse mates. > - if data is single ended, each element of the collection will be a single [fastq](https://en.wikipedia.org/wiki/FASTQ_format) dataset. ~~Both tools have the same initial Help section but the data is organized a bit differently between the two types of output! The **collection folder shape** is written on the collection folder in your history.~~ _updated_ These are the main differences: ### Download and Extract Reads in FASTQ format from NCBI SRA (current versions) and Faster Download and Extract Reads in FASTQ format from NCBI SRA Collection folder shape: **list of pairs** **List of pairs collections** are a _nested_ listing of files. - Each sample will be written into a grouped element representing that sample. The dataset files will be inside of it. - If the data is single end, each sample will have one fastq file. - If the data is paired end, each sample will have two fastq files. ### ⚠ Download and Extract Reads in FASTQ format from NCBI SRA (legacy versions) Collection folder shape: **list** 🛠\* This is an update! Current versions of both tools **will work the same** at the UseGalaxy servers. Other servers may still host the legacy version.\* **List collections** are a flat listing of files. - Each sample will be written into a single element of a **list collection**. - If the data is single end, each sample will have one fastq file of the single reads. - One file of R1 reads per sample. - If the data is paired end, each sample will be a fastq file containing _both reads together_. - One file with both the R1 and R2 reads per sample. - This is the “interleaved” type of fastq file – many tools will expect that these will be split out into the forward and reverse reads. The tools from the **Seqtk** tool group are one way to do this. * * * So, that is a lot of information! But maybe helps to explain why data in “a collection” might not be enough when choosing how to send data to tools. The datatype matters, but also the shape of the data. You are never stuck in any particular shape! See Collection Operation tools for more. I see you are using the first. The collection folder shape **list** is accepted by **FastQC**. You should be able to connect this to tool. However, because the downstream tools are connected first and this can lead to some workflow metadata conflicts. If you hover over the connection, you will get a message about the “shape” of the collection not being a match for what is expected. 🤖 If there is more than one sample in your list, remember to include **Collection Operations → Flatten Collection** to combine the sample name with the read end label into a unique label (element identifiers) → _sample\_forward_ (single end) or _sample\_forward_ and _sample\_reverse_ (paired end). This will give each **fastq dataset’s report** a unique label when these are later combined with a tool like **MultiQC**. **Try this:** - disconnect all the workflow connection noodles - then, connect the tools again in the _order of execution_ - for your use case, you will be connecting just one of the outputs from the **Download and Extract Reads** – the **single end list connection** - once connected, this informs each downstream tool that a **list collection** of data is passing through - you might need to adjust some of the input parameters on the other tools, too - this is an excellent tip to remind students about! If the noodle don’t connect, reset the workflow metadata by disconnecting all, then connecting from the start. I usually say that getting the inputs on the canvas as the first step is a good idea! **Please give that a try and let us know if it helps or not!** You are also welcome to share back your workflow, or a failed workflow invocation, and we can troubleshoot this more if simply reconnecting is not enough. Maybe one of the downstream tools need an input choice adjusted. Thanks! 🚀 #### BONUS 1 For an example of a similar QA workflow that can handle a paired collection without running into duplicated sample identifiers with FastQC/MultiQC, please see this post. The example workflow could be adjusted to work with single end reads too! This one also has some simple **subworkflows** that might be good for followup for intermediate students. - [Quality Control Start Here! multQC issue and guidance?](https://help.galaxyproject.org/t/quality-control-start-here-multqc-issue-and-guidance/14914) > [@Quality Control Start Here! multQC issue and guidance?](https://help.galaxyproject.org/t/quality-control-start-here-multqc-issue-and-guidance/14914/2): > > A demonstration is in the **Quality Control Q20-L20** public workflow. > How to find it → > > > [@Trimming with different parameters/tools give vastly different end results.](https://help.galaxyproject.org/t/trimming-with-different-parameters-tools-give-vastly-different-end-results/14815/2): > > > > - Search here [GalaxyProject.org](http://GalaxyProject.org) → [GTN Pan-Galactic Workflow Search](https://training.galaxyproject.org/training-material/workflows/list) > > - With the term `Quality Control Q20-L20` Then, this is one discussion from last week where we went into more detail about why this organization works! → [Issues with receiving results from FastQC in MultiQC from several collections of samples - #5 by jennaj](https://help.galaxyproject.org/t/issues-with-receiving-results-from-fastqc-in-multiqc-from-several-collections-of-samples/16397/5) #### BONUS 2 One tip I might suggest is to add in another input! This could isolate the accession input to a single field on the workflow runtime form (at the top!), or a separate text file with a list of accessions (selected from the history). This could be annotated with a usage warning “Single end samples only!” but stated a bit nicer! 🧑‍🔬 ![workflow-passing-text-input](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/5/59512551a5e492bf795d05f98f136e053c484d31.png) . ---
**Author:** ![erdavenport](https://avatars.discourse-cdn.com/v4/letter/e/df788c/32.png) [@erdavenport](https://help.galaxyproject.org/u/erdavenport)\ **Post date:** [October 27, 2025, 5:57pm UTC](https://help.galaxyproject.org/t/download-and-extract-reads-in-fastq-format-from-ncbi-sra-in-workflow-issue/16430/3 "2025-10-27T17:57:34Z")
Thanks for your quick reply @jennaj! So I understand that I have a flat list in the Single-end data, however, it doesn’t look like FastQC accepts that as an input data type: ![Screenshot 2025-10-27 at 1.50.15 PM](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/a/a931b82384bdfd171f36ec0151c52278fbe204bf.png) How can I get FastQC to work off of that list? Sorry if this is really obvious and I’m just missing it! Thanks, Emily ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [October 27, 2025, 6:56pm UTC](https://help.galaxyproject.org/t/download-and-extract-reads-in-fastq-format-from-ncbi-sra-in-workflow-issue/16430/4 "2025-10-27T18:56:23Z")
Glad that helped! For the input area on a tool form, there is an icon for a single dataset, multiple datasets, or a collection. The default is a single dataset. Click on the collection folder to inform the tool to look for a collection in your history. ![selecting-a-collection](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/a/a550aa33a6dcb2df5d926452733c0d8f915b8028.png) If you hover over those icons you see some tool tips! Then, if you still can’t find a dataset, toggle open the accepted formats. The datatype formats the tool is scanning for are listed. Then, when the **shape** of the data _and_ the **format** of the data between your active history and what the tool is expecting are a “match”, your dataset will show up in the listing of potential inputs. This is a bit of automatic QC built into Galaxy to prevent the wrong kind of data from being chosen but can be tricky the first few times! Please give that a try! Based on your screenshot, I would expect the data in collection 2 to show up. More questions are welcome – I’d like to get this working for you! 🙂 ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [October 27, 2025, 8:04pm UTC](https://help.galaxyproject.org/t/download-and-extract-reads-in-fastq-format-from-ncbi-sra-in-workflow-issue/16430/5 "2025-10-27T20:04:28Z")
Hi @erdavenport I had to make an update to my original post but it shouldn’t impact what you are doing! Why? We are in the middle of a release cycle and updates to the way collections are processed has been streamlined. This will not impact what you are doing with single end reads! But I wanted to make sure I put the correct information into this topic for anyone reading it later on. ---
**Author:** ![erdavenport](https://avatars.discourse-cdn.com/v4/letter/e/df788c/32.png) [@erdavenport](https://help.galaxyproject.org/u/erdavenport)\ **Post date:** [October 27, 2025, 8:07pm UTC](https://help.galaxyproject.org/t/download-and-extract-reads-in-fastq-format-from-ncbi-sra-in-workflow-issue/16430/6 "2025-10-27T20:07:31Z")
Ah, ok! Yes, if I select that and reprocess samples, the workflow works as expected. I was also able to go back and adjust the old workflow so that it will work with the lists. Since we already did a bunch of the processing for one sample in class last week, this is what we’ll go with moving forward. Thanks so much for all your rapid help! It’s very much appreciated as I prepped for class tomorrow!