# SortMeRNA not working following the Metatranscriptomic Workflow Tutorial **URL:** \ **Category:** usegalaxy.org support\ **Tags:** troubleshooting, transcriptomics, bg\_sortmerna, humann\ **Created:** [September 23, 2024, 3:48pm UTC](https://help.galaxyproject.org/t/sortmerna-not-working-following-the-metatranscriptomic-workflow-tutorial/13517 "2024-09-23T15:48:28Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![Christopher\_Dutton](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/christopher_dutton/32/6104_2.png) [@Christopher\_Dutton](https://help.galaxyproject.org/u/Christopher_Dutton)\ **Post date:** [September 23, 2024, 3:48pm UTC](https://help.galaxyproject.org/t/sortmerna-not-working-following-the-metatranscriptomic-workflow-tutorial/13517/1 "2024-09-23T15:48:28Z")
Hi! I’ve been going through the tutorial for the Metatranscriptomic Analysis located here - [Hands-on: Metatranscriptomics analysis using microbiome RNA-seq data / Metatranscriptomics analysis using microbiome RNA-seq data / Microbiome](https://training.galaxyproject.org/training-material/topics/microbiome/tutorials/metatranscriptomics/tutorial.html). I’m doing the tutorial on the [Galaxy.org](http://Galaxy.org) server. The SortMeRNA steps execute (do not throw an error) but it does not output anything and says, “The input reads file or reference file is empty, or the reads file is not in FASTA or FASTQ format, no analysis could be made.” So then when HUMAnN runs a few steps later, we get an error and the workflow stops, since HUMAnN needs the output from SortMeRNA. The databases appear to be there and the fastq files appear to be in the proper format, as the earlier tutorial steps work fine. Any ideas? Here is my history - [Galaxy](https://usegalaxy.org/u/cldutton/h/main-metatranscriptomics-analysis) Thanks! Chris ---
**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:** [September 23, 2024, 6:05pm UTC](https://help.galaxyproject.org/t/sortmerna-not-working-following-the-metatranscriptomic-workflow-tutorial/13517/2 "2024-09-23T18:05:20Z")
Welcome, @Christopher_Dutton Thank you for sharing your tutorial link and history! Very helpful 🙂 Please see this post for instructions about what to do → [Filter with SortMeRNA option for generating statistics file missing - #2 by jennaj](https://help.galaxyproject.org/t/filter-with-sortmerna-option-for-generating-statistics-file-missing/12296/2) ---
**Author:** ![Christopher\_Dutton](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/christopher_dutton/32/6104_2.png) [@Christopher\_Dutton](https://help.galaxyproject.org/u/Christopher_Dutton)\ **Post date:** [September 23, 2024, 8:10pm UTC](https://help.galaxyproject.org/t/sortmerna-not-working-following-the-metatranscriptomic-workflow-tutorial/13517/3 "2024-09-23T20:10:06Z")
Hi jennaj, I’m not sure if that is the problem. I’ve checked and I’m already using 2.1b.6. I’m not just missing the statistics file…SortMeRNA is not finding the sequence data AND/OR the rRNA databases. I’m using the tutorial as it is written, with their two input files, and cannot get past this step due to this issue. I’ve tried this on the other galaxy servers as well, and each has their own issues. Any other thoughts? Thanks for the help. Chris ---
**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:** [September 23, 2024, 9:54pm UTC](https://help.galaxyproject.org/t/sortmerna-not-working-following-the-metatranscriptomic-workflow-tutorial/13517/4 "2024-09-23T21:54:51Z")
Hum, OK, I’l review your history again. ---
**Author:** ![Christopher\_Dutton](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/christopher_dutton/32/6104_2.png) [@Christopher\_Dutton](https://help.galaxyproject.org/u/Christopher_Dutton)\ **Post date:** [September 24, 2024, 2:34pm UTC](https://help.galaxyproject.org/t/sortmerna-not-working-following-the-metatranscriptomic-workflow-tutorial/13517/5 "2024-09-24T14:34:58Z")
Hi Jennaj, I’ve been playing with it some more and keep getting the same result. I’ve tried all the different versions available for SortMeRNA and none of them work with this tutorial anymore. I’m not sure what update broke it? I continue to get the message, _“The input reads file or reference file is empty, or the reads file is not in FASTA or FASTQ format, no analysis could be made”_ And to be clear, I’m doing the stock tutorial on the website with their input files. I’ve made no changes. Any advice/tips you have would be appreciated. Thanks, chris ---
**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:** [September 24, 2024, 5:12pm UTC](https://help.galaxyproject.org/t/sortmerna-not-working-following-the-metatranscriptomic-workflow-tutorial/13517/6 "2024-09-24T17:12:11Z")
Hi @Christopher_Dutton Yes, I found the same result, but only at [UseGalaxy.org](http://UseGalaxy.org). The workflow runs at [UseGalaxy.eu](http://UseGalaxy.eu). Try running it there until we get this corrected. Tracking ticket → [Configuration issue with SortMeRNA at org? · Issue #849 · galaxyproject/usegalaxy-tools · GitHub](https://github.com/galaxyproject/usegalaxy-tools/issues/849) Thanks for reporting the problem, and for all the followup! Very helpful! 🙂