# How to add SILVA\_138.2 latets as a reference to align.seqs tool in GALAXY Server? **URL:** \ **Category:** usegalaxy.org support\ **Tags:** metagenomics\ **Created:** [October 4, 2024, 11:36am UTC](https://help.galaxyproject.org/t/how-to-add-silva-138-2-latets-as-a-reference-to-align-seqs-tool-in-galaxy-server/13597 "2024-10-04T11:36:58Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![Ahmer](https://avatars.discourse-cdn.com/v4/letter/a/a3d4f5/32.png) [@Ahmer](https://help.galaxyproject.org/u/Ahmer)\ **Post date:** [October 4, 2024, 11:36am UTC](https://help.galaxyproject.org/t/how-to-add-silva-138-2-latets-as-a-reference-to-align-seqs-tool-in-galaxy-server/13597/1 "2024-10-04T11:36:58Z")
Hello, I am doing 16s rRNA (V5-V7 region) metagenomics analysis. I downloaded SILVA\_138.2\_SSURef\_NR99\_tax\_silva.fasta.gz from ([https://www.arb-silva.de/fileadmin/silva\_databases/release\_138\_2/Exports/SILVA\_138.2\_SSURef\_NR99\_tax\_silva.fasta.gz](https://www.arb-silva.de/fileadmin/silva_databases/release_138_2/Exports/SILVA_138.2_SSURef_NR99_tax_silva.fasta.gz)). Uploaded its FASTA file as a reference in align.seqs but getting an error. Can someone help with that? Thanks and regards ---
**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 4, 2024, 4:46pm UTC](https://help.galaxyproject.org/t/how-to-add-silva-138-2-latets-as-a-reference-to-align-seqs-tool-in-galaxy-server/13597/2 "2024-10-04T16:46:51Z")
Welcome, @Ahmer Your reference file is the correct data to use but it must be in an uncompressed format. You can uncompress it using the **convert** function under the pencil icon. [FAQ: Converting the file format](https://training.galaxyproject.org/training-material/faqs/galaxy/datasets_convert_datatype.html). Then, to test if the problem is with the database options, you could grab a copy of the version used with tutorials from here and see how that works. If it fails, then you’ll know the issue is with the other inputs, or possibly the parameters used. Tutorial version of Silva (this is a full release, just not the bleeding-edge brand new release! 🙂 ) → [Hands-on: 16S Microbial Analysis with mothur (extended) / 16S Microbial Analysis with mothur (extended) / Microbiome](https://training.galaxyproject.org/training-material/topics/microbiome/tutorials/mothur-miseq-sop/tutorial.html#hands-on-obtaining-our-data) If that is not enough, what is the error? We can probably help to solve it. How to share your work is in the banner at this forum, or see here directly for the instructions. → [How to get faster help with your question](https://help.galaxyproject.org/docs?topic=11469) Let’s start there, thanks! ---
**Author:** ![Ahmer](https://avatars.discourse-cdn.com/v4/letter/a/a3d4f5/32.png) [@Ahmer](https://help.galaxyproject.org/u/Ahmer)\ **Post date:** [October 7, 2024, 8:46am UTC](https://help.galaxyproject.org/t/how-to-add-silva-138-2-latets-as-a-reference-to-align-seqs-tool-in-galaxy-server/13597/3 "2024-10-07T08:46:31Z")
Hi Jenny, Thank you for your response. I am currently trying to use the reference data file in FASTA format. However, regarding the database option you’re suggesting, it seems to be tailored for the V4 region. Since my data pertains to the V5-V7 regions, there is a difference in base pair sizes. If you still recommend using the V4 region database despite this difference, I am happy to give it a try. Below is the result from the log file output for your reference. \*\ ***mothur \>** **ign.seqs(fasta=fasta.dat,reference=alignment.reference.dat,align=needl** **leman,ksize=8,processors=4)** **Using 4 processors.** **Reading in the alignment.reference.dat template sequences… [ERROR]: template is not aligned, aborting.** **DONE.** **It took 0 to read 0 sequences.** **mothur \> quit** * * * * * * * * * **Detected 1 [ERROR] messages, please review.** Best regards, Ahmer ---
**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 7, 2024, 5:48pm UTC](https://help.galaxyproject.org/t/how-to-add-silva-138-2-latets-as-a-reference-to-align-seqs-tool-in-galaxy-server/13597/4 "2024-10-07T17:48:56Z")
Hi @Ahmer It sounds like you are getting a scientific result, and that is probably because of the difference in your target region needs.You can get the file for the appropriate region, and use the tutorial file as an example of the expected data format. Meaning: get the correct file for scientific reasons and use the example to confirm failures are not due to technical reasons with the format. Bioinformatics tools are picky about format, anywhere, not just in Galaxy! However the original tool expected the inputs, do the same when running through the application. What I noticed at first was that your file was a _compressed fasta_. If you didn’t try using an uncompressed version yet (same format as the example fasta), I would suggest uncompressing your scientifically correct file to create a technically correct file, and trying tool with that next. 🙂 ---
**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:** [November 11, 2024, 6:30pm UTC](https://help.galaxyproject.org/t/how-to-add-silva-138-2-latets-as-a-reference-to-align-seqs-tool-in-galaxy-server/13597/5 "2024-11-11T18:30:18Z")
2 posts were split to a new topic: [Exploring Mothur hits to a SILVA database](https://help.galaxyproject.org/t/exploring-mothur-hits-to-a-silva-database/13876) ---
**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:** [November 11, 2024, 6:30pm UTC](https://help.galaxyproject.org/t/how-to-add-silva-138-2-latets-as-a-reference-to-align-seqs-tool-in-galaxy-server/13597/6 "2024-11-11T18:30:24Z")