# Is there a protocol for miRNA Sequencing? **URL:** \ **Category:** usegalaxy.eu support\ **Tags:** mirna\ **Created:** [January 20, 2022, 10:04am UTC](https://help.galaxyproject.org/t/is-there-a-protocol-for-mirna-sequencing/7363 "2022-01-20T10:04:18Z")\ **Posts on this page:** 4\ **Page:** 1
**Author:** ![LaiaGutierrez](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/laiagutierrez/32/3054_2.png) [@LaiaGutierrez](https://help.galaxyproject.org/u/LaiaGutierrez)\ **Post date:** [January 20, 2022, 10:04am UTC](https://help.galaxyproject.org/t/is-there-a-protocol-for-mirna-sequencing/7363/1 "2022-01-20T10:04:18Z")
Hi, 😄 I am sequencing miRNA and other small RNA. I found a protocol to obtain the counts for smallRNA. However, this involves getting rid of miRNAs. I wanted to know if there is another protocol to obtain the miRNAs. In this protocol for smallRNA they also align the reads to reference sequences from Drosophila. I am working with humans. I would like to know where I can download these annotation and reference files of miRNA and rRNA. Thank you in advanced, Laia ---
**Author:** ![Flow](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/flow/32/211_2.png) [@Flow](https://help.galaxyproject.org/u/Flow)\ **Post date:** [January 20, 2022, 3:02pm UTC](https://help.galaxyproject.org/t/is-there-a-protocol-for-mirna-sequencing/7363/2 "2022-01-20T15:02:05Z")
Dear @LaiaGutierrez, I am not certain, which training material you have followed, but you can potentially get the counts for your miRNA, if you have the annotation file for your miRNAs. I assume you have done something like a feature counting with a bed or gtf/gff file that contained smallRNA regions. Thus if you would replace this file by a file with miRNAs, then you get the counts. You can obtain annotations for dm6 (Drosophila) from [UCSC](https://genome.ucsc.edu/cgi-bin/hgTables?hgsid=1262664387_GfxU4oHFMOZBBTzaLy6k9PRiNELB&clade=insect&org=0&db=0&hgta_group=genes&hgta_track=refSeqComposite&hgta_table=ncbiRefSeq&hgta_regionType=genome&position=&hgta_outputType=primaryTable&hgta_outFileName=) or [Ensembl](http://www.ensembl.org/info/data/ftp/index.html/). Kind regards, Florian ---
**Author:** ![LaiaGutierrez](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/laiagutierrez/32/3054_2.png) [@LaiaGutierrez](https://help.galaxyproject.org/u/LaiaGutierrez)\ **Post date:** [January 20, 2022, 4:36pm UTC](https://help.galaxyproject.org/t/is-there-a-protocol-for-mirna-sequencing/7363/3 "2022-01-20T16:36:25Z")
Thanks @Flow , I still haven’t analyzed the miRNA because I didn’t know where to find the annotation file. I used this tutorial: [Differential abundance testing of small RNAs](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/srna/tutorial.html) I think I have to align my reads to a miRNA reference sequences file. But I am not sure where to find these reference sequences. Is it the same as the annotaton file? In my case I am working with humans. I attach a picture, so you can tell me if this is what I have to download. In the protocol I followed they use an annotation file and two differne reference sequences, one for rRNA and one for miRNA. I don’t know where to find this. I also don’t know what exactly is the difference between these files (BED, GTF/GFF…). I am very new to bioinformatics and I have trouble with some concepts! 😋 Thanks! ![Genome](https://us1.discourse-cdn.com/flex020/uploads/galaxy/original/2X/6/6ec2da1e308f333facd1c45980214c659e7febe5.png) ---
**Author:** ![Flow](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/flow/32/211_2.png) [@Flow](https://help.galaxyproject.org/u/Flow)\ **Post date:** [January 25, 2022, 12:29pm UTC](https://help.galaxyproject.org/t/is-there-a-protocol-for-mirna-sequencing/7363/4 "2022-01-25T12:29:30Z")
Dear @LaiaGutierrez, > I think I have to align my reads to a miRNA reference sequences file. Yes and no. You have three possibilities. (A) Map only to the reference genome. (B) Map only to the miRNA sequences from a known database. (C) Combine A & B. Option (B) Relies on the database; thus it is quicker, but it might give a misleading count because of multi-mapped reads, and it does not cover unknown miRNA transcripts. I am not 100% familiar with the detailed differences but maybe a combination (option C) is a better approach in your case for human, as mentioned in this [article](https://www.sciencedirect.com/science/article/pii/S266591312030131X). > But I am not sure where to find these reference sequences. One of the biggest miRNA databases is [miRBase](https://www.mirbase.org/). > Is it the same as the annotaton file? No. A reference file for alignemnt is typically in FASTA format. An annotation file usually is in gtf/gff and used, e.g., for counting and an overlap analysis. > In my case I am working with humans. I attach a picture, so you can tell me if this is what I have to download. In the protocol I followed they use an annotation file and two differne reference sequences, one for rRNA and one for miRNA. I don’t know where to find this. I think for human it is better to go to Ensembl and download the file **Gene sets** , which you can filter for miRNAs. > I also don’t know what exactly is the difference between these files (BED, GTF/GFF…). You can find a description of various file formats [here](https://genome.ucsc.edu/FAQ/FAQformat.html). Cheers, Florian