# HISAT2 alignment results in very low in number of mapped reads **URL:** \ **Category:** usegalaxy.org support\ **Tags:** custom-genome, troubleshooting, mapping\ **Created:** [January 27, 2021, 4:59pm UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286 "2021-01-27T16:59:52Z")\ **Posts on this page:** 8\ **Page:** 1
**Author:** ![poojamuk19](https://avatars.discourse-cdn.com/v4/letter/p/eada6e/32.png) [@poojamuk19](https://help.galaxyproject.org/u/poojamuk19)\ **Post date:** [January 27, 2021, 4:59pm UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286/1 "2021-01-27T16:59:52Z")
Hi all I am trying to analyse my mRNAseq data using the galaxy reference based workflow ([Hands-on: Hands-on: Reference-based RNA-Seq data analysis / Transcriptomics](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/ref-based/tutorial.html#counting-the-number-of-reads-per-annotated-gene)). I am very new to data analysis and also to galaxy. I tried two different approaches to align my data: 1)RNA-STAR followed by feature counts: In this case the feature counts showed 0 counts. 2) Next, I tried HISAT2 alignment…and that showed just 177 reads… I don’t understand why the read number is so low…do you have any suggestions? ---
**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:** [January 27, 2021, 9:59pm UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286/2 "2021-01-27T21:59:44Z")
Hi @poojamuk19, which reference genome and annotation file are you using? Regards ---
**Author:** ![pshtiwan\_babane](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/pshtiwan_babane/32/2112_2.png) [@pshtiwan\_babane](https://help.galaxyproject.org/u/pshtiwan_babane)\ **Post date:** [February 3, 2021, 6:04pm UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286/3 "2021-02-03T18:04:00Z")
Hi @gallardoalba I have same problem. just few reads align to ref genome. my organism`s ref genome is not supported by galaxy. So, I uploaded fasta file to the history then did alignment using hisat2 and star too. i download fasta and gtf file from here. [Index of /genomes/refseq/bacteria/Streptomyces\_coelicolor/latest\_assembly\_versions](https://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Streptomyces_coelicolor/latest_assembly_versions/) After quality control (trimming) the reads are 32 bp and it is ribo seq data. Many thanks ---
**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:** [February 4, 2021, 12:35am UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286/4 "2021-02-04T00:35:04Z")
Hi @pshtiwan_babane Check to make sure that your inputs are correctly formatted, are a match with each other, and tool parameters that make use of attributes in the GTF are actually present in your GTF dataset. You may also need to do some more QA on your reads. FAQs: [Galaxy Support](https://galaxyproject.org/support/) - [Preparing and using a Custom Reference Genome or Build](https://galaxyproject.org/learn/custom-genomes/) - [Mismatched Chromosome identifiers (and how to avoid them)](https://galaxyproject.org/support/chrom-identifiers/) - [Extended Help for Differential Expression Analysis Tools](https://galaxyproject.org/support/diff-expression/) – useful tips, even if performing other types of analysis GTN Tutorials: [https://training.galaxyproject.org](https://training.galaxyproject.org) - Many tutorials cover read QA/QC steps specific to different analysis goals/tools - This one is a good place to start for basics: [NGS data logistics](https://training.galaxyproject.org/training-material/topics/introduction/tutorials/galaxy-intro-ngs-data-managment/tutorial.html) I also added a few tags to your topic that link to prior Q&A that cover the above items in the context of other people’s analysis. Thanks! ---
**Author:** ![pshtiwan\_babane](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/pshtiwan_babane/32/2112_2.png) [@pshtiwan\_babane](https://help.galaxyproject.org/u/pshtiwan_babane)\ **Post date:** [February 16, 2021, 3:32pm UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286/5 "2021-02-16T15:32:38Z")
hi @gallardoalba I am sorry I can`t do it. must I imported fasta file (ref genome) through ftp or is it ok i uploaded it. then I used NormalizeFasta still doesn’t work! kindly, can you help me more? Many 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 17, 2021, 10:46am UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286/6 "2021-02-17T10:46:05Z")
Hi @pshtiwan_babane, I’ll reproduce the analysis by using [that datasets](https://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Streptomyces_coelicolor/latest_assembly_versions/GCF_013363625.1_ASM1336362v1/). I’ll inform you as soon as I get some results. Regards ---
**Author:** ![pshtiwan\_babane](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/pshtiwan_babane/32/2112_2.png) [@pshtiwan\_babane](https://help.galaxyproject.org/u/pshtiwan_babane)\ **Post date:** [February 17, 2021, 7:40pm UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286/7 "2021-02-17T19:40:29Z")
Hi @gallardoalba This will be awesome. Really appreciate your help . ---
**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:** [December 20, 2023, 11:59pm UTC](https://help.galaxyproject.org/t/hisat2-alignment-results-in-very-low-in-number-of-mapped-reads/5286/9 "2023-12-20T23:59:19Z")
Closed on December 20, 2023, 11:59pm UTC