FMAP

Functional Mapping and Analysis Pipeline for metagenomics and metatranscriptomics studies

Some example results are available at the homepage: https://qbrc.swmed.edu/FMAP/.

Features

FMAP provides a more sensible reference protein sequence database based on UniRef.
Identification of differentially-abundant genes KEGG Orthology
Mapping differentially-abundant genes to pathways and modules (KEGG Pathway and KEGG Module)
Mapping differentially-abundant genes to operons (ODB (v3))

Requirements

Perl - scripting language
R - statistical computing

Statistics::R - Perl interface with the R statistical program

Use CPAN to install the module
```
perl -MCPAN -e 'install Statistics::R'
```

or download the source and compile manually

wget 'http://search.cpan.org/CPAN/authors/id/F/FA/FANGLY/Statistics-R-0.34.tar.gz'
tar zxf Statistics-R-0.33.tar.gz
cd Statistics-R-0.33
perl Makefile.PL
make
make test
make install

Mapping program providing BLASTX search of sequencing reads: DIAMOND or USEARCH
Linux commands: wget, cat, sort
Bio::DB::Taxonomy - Access to a taxonomy database (which is required only if you want to build a custom database.)
XML::LibXML - Perl Binding for libxml2 (which is required only if you want to download genome sequences.)

Command usages

FMAP_database.pl
- Process
- Input
  1. UniRef sequence identity (50, 90, or 100)
  2. (optional) NCBI taxonomy IDs (integer)
- Require Bio::DB::Taxonomy.
- The following data files will be downloaded through FTP connection. If you have a problem in the FTP connection, please download the files through another method and copy them into "FMAP_data" directory before executing "FMAP_database.pl" command.
  ftp://ftp.ncbi.nlm.nih.gov/pub/taxonomy/taxdump.tar.gz
  ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/idmapping/idmapping.dat.gz
  ftp://ftp.uniprot.org/pub/databases/uniprot/uniref/uniref100/uniref100.fasta.gz
  or ftp://ftp.uniprot.org/pub/databases/uniprot/uniref/uniref90/uniref90.fasta.gz
  or ftp://ftp.uniprot.org/pub/databases/uniprot/uniref/uniref50/uniref50.fasta.gz
- Require HTTP connection for KEGG API.
```
Usage:   perl FMAP_database.pl [options] 50|90|100 [NCBI_TaxID [...]]
```

Options: -h display this help message -s switch database -r redownload data


* **FMAP_prepare.pl**

Usage: perl FMAP_prepare.pl [options]

Options: -h display this help message -r redownload data -m FILE executable file path of mapping program, "diamond" or "usearch" [diamond] -k download prebuilt KEGG files


* **FMAP_assembly.pl**
  * Process
    ![](FMAP_assembly.process.png)
    * Read mapping: nucleotide sequence alignment using [BWA](http://bio-bwa.sourceforge.net)
    * ORF mapping: protein sequence alignment using [DIAMOND](http://ab.inf.uni-tuebingen.de/software/diamond/)
  * Input
    1. Prefix of output files
    2. De novo assembled sequences in FASTA format
       * A FASTA file can be generated by metagenome assemblers such as [SPAdes](http://cab.spbu.ru/software/spades/) and [MetaVelvet](http://metavelvet.dna.bio.keio.ac.jp).
       * A FASTA file containing target genome sequences can be input instead.
    3. Whole metagenomic/metatranscriptomic shotgun sequencing reads in FASTQ or FASTA format
       * Multiple read files can be specified.
       * Paired-end read files must be specified comma-separated like "input.R1.fastq,input.R2.fastq".
       * The read files can be compressed by gzip.
  * Output
    1. Prefix.region.abundance.txt (abundances of ORF regions mapping to KEGG orthologies)
    2. Prefix.abundance.txt (abundances of KEGG orthologies)

Usage: perl FMAP_assembly.pl [options] output.prefix assembly.fasta [input.fastq|input.R1.fastq,input.R2.fastq [...]] > summary.txt

Options: -h display this help message -A STR prepared assembly prefix -B input indexed sorted BAM file instead of FASTQ file -m FILE executable file path of mapping program, "diamond" or "usearch" [diamond] -p INT number of threads [1] -e FLOAT maximum e-value to report alignments [10] -t DIR directory for temporary files [$TMPDIR or /tmp] -a FLOAT search acceleration for ublast [0.5] -C STR codon and translation e.g. ATG=M [NCBI genetic code 11 (Bacterial, Archaeal and Plant Plastid)] -S STR comma-separated start codons [GTG,ATG,CTG,TTG,ATA,ATC,ATT] -T STR comma-separated termination codons [TAG,TAA,TGA] -l INT minimum translation length [10] -c FLOAT minimum coverage [0.8] -q INT minimum mapping quality [0] -s STR strand specificity, "f" or "r" -P STR contig prefix used for abundance estimation


* **FMAP_assembly_centrifuge.pl**
  * Require [Centrifuge](https://ccb.jhu.edu/software/centrifuge/).
  * Input
    1. FMAP_assembly.region.txt (ORF regions mapping to KEGG orthologies generated by FMAP_assembly)
    2. De novo assembled sequences in FASTA format
    3. Centrifuge index filename prefix (minus trailing .X.cf)
  * Output: FMAP_assembly.region.taxon.txt (FMAP_assembly.region.txt including a column of [NCBI taxonomy](https://www.ncbi.nlm.nih.gov/taxonomy) IDs (integer))

Usage: perl FMAP_assembly_taxon.pl [options] FMAP_assembly.region.txt assembly.fasta centrifuge.index

Options: -h display this help message -p INT number of threads [1]


* **FMAP_assembly_heatmap.pl**
  * Require [Bio::DB::Taxonomy](http://search.cpan.org/dist/BioPerl/Bio/DB/Taxonomy.pm).
  * Input: FMAP_assembly.abundance.txt (abundances generated by FMAP_assembly)
  * Output: HTML format of abundance heatmap table

Usage: perl FMAP_assembly_heatmap.pl [options] [name=]FMAP_assembly.abundance.txt [...] > FMAP_assembly_heatmap.html

Options: -h display this help message -c FILE comparison output file including orthology and filter columns -f INT HTML font size -w INT HTML table cell width


* **FMAP_assembly_operon.pl**
  * Input: FMAP_assembly.region.txt (ORF regions mapping to KEGG orthologies generated by FMAP_assembly)
  * Output: FMAP_assembly_operon.txt ([ODB (v3)](http://operondb.jp) known operons consisting of orthologies located together on an assembled contig/scaffold/transcript)

Usage: perl FMAP_assembly_operon.pl [options] FMAP_assembly.region.txt > FMAP_assembly_operon.txt

Options: -h display this help message -a print single-gene operons as well


* **FMAP_download_genome.pl**
  * Input: [NCBI taxonomy](https://www.ncbi.nlm.nih.gov/taxonomy) IDs (integer)
  * Output: FASTA file containing genome sequences
  * Require [XML::LibXML](http://search.cpan.org/dist/XML-LibXML/LibXML.pod).

Usage: perl FMAP_download_genome.pl [options] NCBI_TaxID [...] > genome.fasta

Options: -h display this help message -a assembly instead of genome


* **FMAP_download.pl**

Usage: perl FMAP_download.pl [options]

Options: -h display this help message -m FILE executable file path of mapping program, "diamond" or "usearch" [diamond] -k download prebuilt KEGG files -x download only KEGG files


* **FMAP_mapping.pl**
  * Input: whole metagenomic (or metatranscriptomic) shotgun sequencing reads in FASTQ or FASTA format
  * Output: best-match hits in NCBI BLAST ‑m8 (= NCBI BLAST+ ‑outfmt 6) format

Usage: perl FMAP_mapping.pl [options] input1.fastq|input1.fasta [input2.fastq|input2.fasta [...]] > blastx_hits.txt

Options: -h display this help message -m FILE executable file path of mapping program, "diamond" or "usearch" [diamond] -p INT number of threads [1] -e FLOAT maximum e-value to report alignments [10] -t DIR directory for temporary files [$TMPDIR or /tmp] -a FLOAT search acceleration for ublast [0.5]


* **FMAP_quantification.pl**
  * Input: output of "FMAP_mapping.pl"
  * Output: abundances (RPKM) of KEGG orthologies
  * Output columns: [KEGG Orthology](http://www.genome.jp/kegg/ko.html) ID, orthology definition, abundance (RPKM)

Usage: perl FMAP_quantification.pl [options] blast_hits1.txt [blast_hits2.txt [...]] > abundance.txt

Options: -h display this help message -c use CPM values instead of RPKM values -i FLOAT minimum percent identity [80] -l FILE tab-delimited text file with the first column having protein names and the second column having the sequence lengths -o FILE tab-delimited text file with the first column having protein names and the second column having the orthology names -d FILE tab-delimited text file with the first column having orthology names and the second column having the definitions -w FILE tab-delimited text file with the first column having read names and the second column having the weights


* **FMAP_table.pl**
  * Input: outputs of "FMAP_quantification.pl"
  * Output: abundance table
  * Output columns: [KEGG Orthology](http://www.genome.jp/kegg/ko.html) ID, orthology definition, abundance of sample1, abundance of sample2, ...

Usage: perl FMAP_table.pl [options] [name1=]abundance1.txt [[name2=]abundance2.txt [...]] > abundance_table.txt

Options: -h display this help message -c use raw read counts (readCount|count) instead of RPKM values -d use normalized mean depths (meanDepth/genome) instead of RPKM values -f use fractions -n do not print definitions -r print ORF regions


* **FMAP_comparison.pl**
  * Input: output of "FMAP_table.pl", sample group information
  * Output: comparison test statistics for orthologies
  * Output columns: [KEGG Orthology](http://www.genome.jp/kegg/ko.html) ID, orthology definition, log2 fold change, p-value, FDR-adjusted p-value, filter (pass or fail)

Usage: perl FMAP_comparison.pl [options] abundance_table.txt control1[,control2[...]] case1[,case2[...]] [...] > orthology_test_stat.txt

Options: -h display this help message -t STR statistical test for comparing sample groups, "kruskal", "anova", "poisson", "quasipoisson", "metagenomeSeq" [kruskal] -f FLOAT fold change cutoff [2] -p FLOAT p-value cutoff [0.05] -a FLOAT FDR-adjusted p-value cutoff [1]


* **FMAP_pathway.pl**
  * Input: output of "FMAP_comparison.pl"
  * Output: pathways enriched in filter-passed orthologies
  * Output columns: [KEGG Pathway](http://www.genome.jp/kegg/pathway.html) ID, pathway definition, orthology count, coverage, p-value, [KEGG Orthology](http://www.genome.jp/kegg/ko.html) IDs with colors
  * [KEGG Orthology](http://www.genome.jp/kegg/ko.html) IDs with colors: input of [KEGG Pathway](http://www.genome.jp/kegg/pathway.html) mapping (http://www.kegg.jp/kegg/tool/map_pathway2.html)

Usage: perl FMAP_pathway.pl [options] orthology_test_stat.txt > pathway.txt

Options: -h display this help message


* **FMAP_module.pl**
  * Input: output of "FMAP_comparison.pl"
  * Output: modules enriched in filter-passed orthologies
  * Output columns: [KEGG Module](http://www.genome.jp/kegg/module.html) ID, module definition, orthology count, coverage, p-value, [KEGG Orthology](http://www.genome.jp/kegg/ko.html) IDs with colors
  * [KEGG Orthology](http://www.genome.jp/kegg/ko.html) IDs with colors: input of [KEGG Pathway](http://www.genome.jp/kegg/pathway.html) mapping (http://www.kegg.jp/kegg/tool/map_pathway2.html)

Usage: perl FMAP_module.pl [options] orthology_test_stat.txt > module.txt

Options: -h display this help message


* **FMAP_operon.pl**
  * Input: output of "FMAP_comparison.pl"
  * Output: operons consisting of filter-passed orthologies
  * Output columns: [ODB (v3)](http://operondb.jp) known operon IDs, operon definition, log2 fold change, [KEGG Orthology](http://www.genome.jp/kegg/ko.html) IDs, [KEGG Pathway](http://www.genome.jp/kegg/pathway.html) IDs

Usage: perl FMAP_operon.pl [options] orthology_test_stat.txt > operon.txt

Options: -h display this help message -a print single-gene operons as well


* **FMAP_plot.pl**
  * Input: output of "FMAP_pathway.pl", "FMAP_module.pl", or "FMAP_operon.pl"
  * Output: PNG format image file of p-value plot

Usage: perl FMAP_plot.pl [options] pathway.txt|module.txt|operon.txt plot.pdf

Options: -h display this help message -w INT plot width [12] -h INT plot height [8] -l FLOAT plot left margin [20] -p FLOAT p-value cutoff [0.05] -c FLOAT coverage cutoff [0 for pathway, 1 for module and operons] -d do not print definition


* **FMAP_all.pl**
  * Input: configuration table file
  * Input columns: group (control, ...), sample name, input file of "FMAP_mapping.pl"
  * Output: script file including all FMAP commands, all FMAP outputs

Usage: perl FMAP_all.pl [options] input.config [output_prefix]

Options: -h display this help message -s generate a script, but not execute it -m FILE mapping: executable file path of mapping program, "diamond" or "usearch" [diamond] -t INT mapping: number of threads [1] -c STR comparison: statistical test for comparing sample groups, "kruskal", "anova", "poisson", "quasipoisson", "metagenomeSeq" [kruskal] -f FLOAT comparison: fold change cutoff [2] -p FLOAT comparison: p-value cutoff [0.05] -a FLOAT comparison: FDR-adjusted p-value cutoff [1]



## Command orders

* Use the prebuilt database (UniRef90 and bacteria/archaea/fungi)
  1. FMAP_download.pl
  2. FMAP_mapping.pl
  3. FMAP_quantification.pl
  4. FMAP_table.pl
  5. FMAP_comparison.pl
  6. FMAP_pathway.pl
  7. FMAP_module.pl
  8. FMAP_operon.pl

* Use a custom database (you can define UniRef and taxonomy.)
  1. FMAP_database.pl
  2. FMAP_prepare.pl
  3. FMAP_mapping.pl
  4. FMAP_quantification.pl
  5. FMAP_table.pl
  6. FMAP_comparison.pl
  7. FMAP_pathway.pl
  8. FMAP_module.pl
  9. FMAP_operon.pl

## Citation

Kim J, Kim MS, Koh AY, Xie Y, Zhan X.
"FMAP: Functional Mapping and Analysis Pipeline for metagenomics and metatranscriptomics studies"
BMC Bioinformatics. 2016 Oct 10;17(1):420.
PMID: [27724866](https://www.ncbi.nlm.nih.gov/pubmed/27724866)

jiwoongbio / FMAP

readme

FMAP

Features

Requirements

Command usages