sMETASeq

Purpose

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

sMETASeq command line pipeline

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. This command line based pipeline requires installation of several tools in addition to kraken2 and its database. We recommend install these dependencies using conda. Conda can be installed on Mac and Linux as shown below.

Install conda:

Mac: https://docs.conda.io/projects/conda/en/latest/user-guide/install/macos.html` Linux: https://docs.conda.io/projects/conda/en/latest/user-guide/install/linux.html

Make directory in which scripts and other dependencies will be stored and create a conda environment

mkdir sMETASeq
cd sMETASeq
conda create --name sMETASeq

When conda asks you to proceed, type y

To activate this environment, use

 conda activate sMETASeq

To deactivate an active environment, use

 conda deactivate

To activate the environment you might have to type bash and conda activate sMETASeq

Install dependencies using conda:

conda install -c bioconda kraken2
conda install -c bioconda htseq
conda install -c bioconda bowtie2
conda install -c bioconda cutadapt

Download pre-compiled kraken2 standard database:

wget -r --no-parent -nH --cut-dirs=2 -R "index.html*" http://havpryd.medisin.ntnu.no/robinm/sMETASeq/kraken2_DB/

Download and unzip bowtie2 index:

wget ftp://ftp.ncbi.nlm.nih.gov/genomes/archive/old_genbank/Eukaryotes/vertebrates_mammals/Homo_sapiens/GRCh38/seqs_for_alignment_pipelines/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.tar.gz

tar -xvzf GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.tar.gz

Download miRBase and RNACentral databases:

wget ftp://mirbase.org/pub/mirbase/CURRENT/genomes/hsa.gff3

wget ftp://ftp.ebi.ac.uk/pub/databases/RNAcentral/releases/14.0/genome_coordinates/gff3/homo_sapiens.GRCh38.gff3.gz

gunzip homo_sapiens.GRCh38.gff3.gz

Modify RNACentral file by adding "chr"-prefix:

awk '{ if($1 !~ /^#/){print "chr"$0} else{print $0} }' homo_sapiens.GRCh38.gff3 > homo_sapiens.GRCh38.chr.gff3

Download example data and sMETASeq scripts

Edit: Sample data has been removed. Please email me: [email protected] or [email protected]

wget -r --no-parent -nH --cut-dirs=3 -R "index.html*" http://havpryd.medisin.ntnu.no/robinm/sMETASeq/ExampleData/
wget -r --no-parent -nH --cut-dirs=3 -R "index.html*" http://havpryd.medisin.ntnu.no/robinm/sMETASeq/scripts/

Kraken databases can be downloaded from here:

 https://benlangmead.github.io/aws-indexes/k2

Running sMETASeq on example data

Depending on which library preparation method was used to create the small RNA libraries, we have created three different bash-scripts: sMETASeq_TruSeq.sh sMETASeq_NEXTFlex.sh sMETASeq_NEBNext.sh. They differ in how adapters are being trimmed from the sequences. In our example data, we will use sMETASeq_NEXTFlex.sh. The input files are specified in files.txt. The file names should be the string preceding .fastq.gz of the filename.

The scripts are run by typing sh sMETASeq_NEXTFlex.sh

The pipeline will create three output files for each input file:

$FILE.kraken_report.csv # The kraken2 output file containing counts to each OTU in kraken

$FILE.miRBase.csv # Counts for mature miRNAs from miRBase

$FILE.RNACentral.csv # Counts for ncRNAs from RNACentral

Output results for the the example data can be sent via fpt upon request: Please email me: [email protected] or [email protected]