metaRUpore

metaRUpore codifies the analytical procedures involved in our 2-stage selective sequencing based community normalization workflow. The workflow is aimed to normalize the genome coverage of different populations within a community by unblocking reads of the dominant populations during nanopore selective sequencing. The metagenome (fasta format) generated in the first stage regular sequencing (~1hour) will be used as input to determine the target to unblock duing the 2nd-stage selective sequencing (48hour).

metaRUpore will generated a reference.fasta, a target list as well as a toml file required to implement 2nd-stage selective sequencing with ReadFish(https://github.com/LooseLab/readfish).

Please read below instructions carefully to avoid unnecessary errors.

Installation

Pre-requisites for ARGpore

GNU parallel	### sudo apt install parallel
git lfs	        ### sudo apt install git-lfs
fgrep
R and library: plyr, data.table, doParallel, foreach

Setup metaRUpore

git clone https://github.com/sustc-xylab/metaRUpore.git

cd metaRUpore

bash ./setup.sh

The setup.sh will install Centrifuge and then download bacteria+archaea+virus database for Centrifuge, MetaPhlan2 Markergene for you. It will take at least 1 hour to finish, please stay patient :)

Using MetaRUpore

Once installed metaRUpore, all needed analysis is wrapped up in one executable named metaRUpore.sh. Please use bash instead of sh to initiate metaRUpore.sh

First of all, the user should fill in the taxa names they intended to manually set to accept during selective sequencing into the keep.csv ,this keep.csv should be kept in the same directory of your input.fasta

Since metaRUpore will read keep.csv by column, therefore you don't need to keep the hiearchy phylogeny structure of your inputed taxon. Instead simply input the taxon names you don't want to eject in the correct taxonomic level.

This is how keep.csv looks like:

|kingdom |phylum|class|order|family|genus|species| | --------| ---- | | Archaea | your input | | Virus | your input |

NOTICE: To avoid cross-writing of intermediate files, each metaRUpore run should have an independent working directory. To improve annotation accuracy, metaRUpore only analyze the > 1kbp portion of your input.fasta.

Recommended usage of metaRUpore is:

mkdir -p demo
cp keep.csv demo
cp test.fa demo 
cd demo 
bash $PATH_to_metaRUpore/metaRUpore.sh -f test.fa -t 20 > metaRUpore.log

Output files

All output files of metaRUpore are stored in the working directory.

Main output files include:

reference.fa    a fasta file containing reads that could be used as reference for the ReadFish selective sequencing run
target.txt      a list of reads name that wll be used as target for ReadFish selective sequencing (reads hit to target will be ejected during selective sequencing)
metaRU.toml     toml file containing the configuration of ReadFish selective sequencing run

The taxonomy annotation algorithm of metaRUpore is the sampe with our ARGpore2 tool (https://github.com/sustc-xylab/ARGpore2), in which taxonomic annotation is derived by combining results of a modified version of Centrifuge and MetaPhlan2 markergene database.

Citation:

If you use metaRUpore in your metagenome analysis please cite: to be filled

Tools included in metaRUpore should be also cited, these tools includes:

last, Centrifuge, MetaPhlan2, GNU parallel, R, python and relavent packages

colindaven / metaRUpore