FUNpore

FUNpore is designed to do frame-shift correction of nanopore 1D/2D reads (fasta format) and then carried out taxonomic annotation of the corrected reads with taxator-tk and KRAKEN. Please read this manual carefully to avoid unnecessary errors for FUNpore implementation.

Author: Yu XIA

Email: shuixia100@gmail.com

version 1.0

Installation

Pre-requisites for FUNpore

ruby 2.3.1p112
python2.7
samtools 1.9
R and library: foreach, doParallel, seqinr, plyr, data.table, Rsamtools
GNU parallel
blast+ 2.8.1+ or higher

Download the lastest FUNpore

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

cd FUNpore

Once download FUNpore package, all needed analysis is wrapped up in one executable named FUNpore.sh

The FUNpore_CONFIG contains the PATH for database required for FUNpore, this file should always be stored in the same directory with FUNpore.sh.

Before runing FUNpore, users should modify FUNpore_CONFIG with their specific database PATH

Tools supported out-of-the-box are

lastal, one copy of last-938 included in FUNpore package
taxator-tk, one copy of taxator-tk included in FUNpore package
KRAKEN, one copy of KRAKEN 0.10.6-unreleased included in FUNpore package

Database to prepare before FUNpore run

Downloading comprehensive database usually takes quite long time. please stay patient :)

** LASTAL database **
we recommend run lastal against NCBI refseq_protein database, you may download the blast-formatted refseq_protein database fron NCBI and then extract fasta from the blast-formatted database downloaded, finally build last index with refseq_protein
	
	1. download the blast-formatted refseq_protein database
	
		perl  $PATH_to_FUNpore/bin/update_blastdb.pl --passive --decompress refseq_protein
	
	2. extracted the fasta from the blast-formatted refseq_protein database
	
		$PATH_to_FUNpore/bin/blastdbcmd -db refseq_protein -dbtype prot -entry all -outfmt "%f"  -out refseq_protein.fa 

	3. build last index with the fasta file extracted
	
		$PATH_to_FUNpore/bin/last-938/src/lastdb -Q 0 -P 50 -p -cR01 -v refseq_protein_lastdb refseq_protein.fa
	
	4. specify the path and name of the last database in FUNpore_CONFIG 
	
** NCBI nt database for BLAST+ and nodes.dmp of corresponding taxonomy ** 
	
	1. download NCBI preformatted nt database with:
		
		perl  $PATH_to_FUNpore/bin/update_blastdb.pl --passive --decompress nt
		
	specify the name of the nt database in FUNpore_CONFIG
	
	2. download NCBI taxonomy with：
		
		wget ftp://ftp.ncbi.nlm.nih.gov/pub/taxonomy/taxdump.tar.gz
	
	Place the files names.dmp and nodes.dmp in a folder and specify its path in FUNpore_CONFIG. keep in mind that the taxonomy files are modified on a regular basis.  
	
** Default database of KRAKEN **
	
	To create the standard Kraken database, you can use the following command:
	
		$PATH_to_FUNpore/bin/libexec/kraken/kraken-build --standard --threads 24 --db $DBNAME
	
	Replace "$DBNAME" with your preferred database name/location. 
	
	specify the path of the KRAKEN database in FUNpore_CONFIG

** Lineage databse converted from NCBI taxonomy ** 
	
	wget https://gitlab.com/zyxue/ncbitax2lin-lineages/blob/master/lineages-2019-02-20.csv.gz
	
	gunzip lineages-2019-02-20.csv.gz
	
	mv lineages-2019-02-20.csv $PATH_to_FUNpore/database

Using FUNpore

There are two steps to run FUNpore as below. Please NOTICE, since FUNpore will overwrite intermediate files from previous run, as a result, each working directory can only be used for ONE instance of FUNpore run. Additionally, please use bash instead of sh to initiate FUNpore.

Try out FUNpore with test.fa

step1 create a working directory for FUNpore run and copy the test.fa to the created directory
	
	mkdir testdir 
	
	cp test.fa testdir 
	
step2 run FUNpore in the directory created, $PATH is the FUNpore installing directory 
	
	cd testdir
	
	bash $PATH_to_FUNpore/FUNpore.sh -f ./test.fa -t 20 -c 200000 > FUNpore.log

Output files

all output files of FUNpore are stored in a directory named $YOUR_TARGET_FASTA_NAME_FUNpore_nowtime, this directory will include:

input_framecorrect.fa 	frame-shift corrected nanopore reads
input_framecorrect.summary.csv	summary of the frame-shift correction 
input_framecorrect.fa_taxa.tab	phylogenetic assignment of frame-shift corrected reads by taxator-tk(nt based homology search) and KRAKEN (kmer search)

Citation:

If you use FUNpore in your nanopore dataset analysis please cite:

Xia, Y., Li, A.-D., Deng, Y., Jiang, X.-T., Li, L.-G., and Zhang, T. (2017) MinION Nanopore Sequencing Enables Correlation between Resistome Phenotype and Genotype of Coliform Bacteria in Municipal Sewage. Front Microbiol 8: 2105.

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

last, blast+, taxator-tk, kraken, samtools, GNU parallel, ruby, R