This tutorial shows how to run sMETASeq using Galaxy(1) (https://usegalaxy.org/)
sMETASeq is an analysis pipeline to detect microbes, including virus, bacteria and other pathogens, from small RNA-seq data. The pipeline will also include steps to generate host small RNA expression profiles. The microbes are detected using the kraken database and the host small RNAs are detected using miRBase and RNACentral, for miRNAs and other small RNAs, respectively.
Additional resources:
Sneak peek of the paper: https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3525549
This tutorial shows step-by-step how to generate a metagenomics and host small RNA count tables from small RNA sequencing data. The output of the pipeline is expression tables that can be further analyzed within a statistical framwork such as R.
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Create Galaxy account (Galaxy europe) and login: https://usegalaxy.eu/login
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Download references files to your local computer and unzip the RNACentral file:
- RNACentral: ftp://ftp.ebi.ac.uk/pub/databases/RNAcentral/releases/14.0/genome_coordinates/gff3/homo_sapiens.GRCh38.gff3.gz
- miRBase: ftp://mirbase.org/pub/mirbase/CURRENT/genomes/hsa.gff3
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If not in fastq.gz format, prepare gzipped fastq files
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Within Galaxy, load fastq.gz files and the two reference files using ">Get Data" and ">Upload File from your computer". For larger files we recommend using FTP upload: https://galaxyproject.org/ftp-upload/ If working in a linux environment, files can be transferred via FTP using the following command:
curl -T {"file1"} ftp://ftp.usegalaxy.eu --user [email protected] --sslIn this example, file1 is transferred. Please change email to your login email. -
Download and import sMETASeq Galaxy workflow
Three different workflows are available depending on which library preparation kit was using to create the sRNA-data. Download and input the desired workflow into your Galaxy environment using the Import tab under Workflow
- TruSeq data: https://usegalaxy.eu/u/robin/w/smetaseq---truseq-input
- NextFlex data: https://usegalaxy.eu/u/robin/w/smetaseq---nextflex-input
- NEBNext data: https://usegalaxy.eu/u/robin/w/smetaseq---nebnext-input
- For each input file, sMETASeq provides three output files:
- miRNA profile: Count data for mature miRNAs
- sncRNA profile: Count data for small RNAs within RNACentral
- Metagenomics profile: Count data for taxonomic labels within the kraken database
Please direct any questions or concerns on the issue site.
(1) Afgan E et al. 2016 The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 44, W3–W10 doi:10.1093/nar/gkw343