MetaPhlAn 2 clade-abundance estimation

The basic usage of MetaPhlAn 2 consists in the identification of the clades (from phyla to species and strains in particular cases) present in the metagenome obtained from a microbiome sample and their relative abundance. This correspond to the default analysis type (--analysis_type rel_ab).

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Profiling a metagenome from raw reads:

metaphlan2 metagenome.fastq --input_type fastq

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You can take advantage of multiple CPUs and save the intermediate BowTie2 output for re-running

MetaPhlAn extremely quickly:
metaphlan2 metagenome.fastq --bowtie2out metagenome.bowtie2.bz2 --nproc 5 --input_type fastq

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If you already mapped your metagenome against the marker DB (using a previous MetaPhlAn run), you can obtain the results in few seconds by using the previously saved --bowtie2out file and specifying the input (--input_type bowtie2out):

metaphlan2 metagenome.bowtie2.bz2 --nproc 5 --input_type bowtie2out

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You can also provide an externally BowTie2-mapped SAM if you specify this format with --input_type. Two steps: first apply BowTie2 and then feed MetaPhlAn2 with the obtained sam:

bowtie2 --sam-no-hd --sam-no-sq --no-unal --very-sensitive -S metagenome.sam -x /usr/share/metaphlan2/db_v20/mpa_v20_m200 -U metagenome.fastq metaphlan2 metagenome.sam --input_type sam > profiled_metagenome.txt

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Multiple alternative ways to pass the input are also available:

cat metagenome.fastq | metaphlan2 --input_type fastq
tar xjf metagenome.tar.bz2 --to-stdout | metaphlan2 --input_type fastq
metaphlan2 --input_type fastq < metagenome.fastq
metaphlan2 --input_type fastq <(bzcat metagenome.fastq.bz2)
metaphlan2 --input_type fastq <(zcat metagenome_1.fastq.gz metagenome_2.fastq.gz)

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We can also natively handle paired-end metagenomes, and, more generally, metagenomes stored in multiple files (but you need to specify the --bowtie2out parameter):

metaphlan2 metagenome_1.fastq,metagenome_2.fastq --bowtie2out metagenome.bowtie2.bz2 --nproc 5 --input_type fastq

MetaPhlAn 2 strain tracking

MetaPhlAn 2 introduces the capability of charachterizing organisms at the strain level using non aggregated marker information. Such capability comes with several slightly different flavours and are a way to perform strain tracking and comparison across multiple samples. Usually, MetaPhlAn 2 is first ran with the default --analysis_type to profile the species present in the community, and then a strain-level profiling can be performed to zoom-in into specific species of interest. This operation can be performed quickly as it exploits the --bowtie2out intermediate file saved during the execution of the default analysis type.

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The following command will output the abundance of each marker with a RPK (reads per kil-base) higher 0.0. (we are assuming that metagenome_outfmt.bz2 has been generated before as shown above).

metaphlan2 -t marker_ab_table metagenome_outfmt.bz2 --input_type bowtie2out > marker_abundance_table.txt

The obtained RPK can be optionally normalized by the total number of reads in the metagenome to guarantee fair comparisons of abundances across samples. The number of reads in the metagenome needs to be passed with the '--nreads' argument

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The list of markers present in the sample can be obtained with '-t marker_pres_table'

metaphlan2 -t marker_pres_table metagenome_outfmt.bz2 --input_type bowtie2out > marker_abundance_table.txt

The --pres_th argument (default 1.0) set the minimum RPK value to consider a marker present

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The list '-t clade_profiles' analysis type reports the same information of '-t marker_ab_table' but the markers are reported on a clade-by-clade basis.

metaphlan2 -t clade_profiles metagenome_outfmt.bz2 --input_type bowtie2out > marker_abundance_table.txt

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Finally, to obtain all markers present for a specific clade and all its subclades, the '-t clade_specific_strain_tracker' should be used. For example, the following command is reporting the presence/absence of the markers for the B. fragulis species and its strains the optional argument --min_ab specifies the minimum clade abundance for reporting the markers

$ metaphlan2 -t clade_specific_strain_tracker --clade s__Bacteroides_fragilis metagenome_outfmt.bz2 --input_type bowtie2out > marker_abundance_table.txt

positional arguments

INPUT_FILE

the input file can be:

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a fastq file containing metagenomic reads

OR

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a BowTie2 produced SAM file.

OR

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an intermediary mapping file of the metagenome generated by a previous MetaPhlAn run

If the input file is missing, the script assumes that the input is provided using the standard input, or named pipes. IMPORTANT: the type of input needs to be specified with --input_type

OUTPUT_FILE

the tab-separated output file of the predicted taxon relative abundances [stdout if not present]

Required arguments

--input_type {fastq,fasta,multifasta,multifastq,bowtie2out,sam}

set whether the input is the multifasta file of metagenomic reads or the SAM file of the mapping of the reads against the MetaPhlAn db. [default 'automatic', i.e. the script will try to guess the input format]

Mapping arguments:

--mpa_pkl MPA_PKL

the metadata pickled MetaPhlAn file

--bowtie2db METAPHLAN_BOWTIE2_DB

The BowTie2 database file of the MetaPhlAn database. Used if --input_type is fastq, fasta, multifasta, or multifastq

--bt2_ps BowTie2 presets

presets options for BowTie2 (applied only when a multifasta file is provided) The choices enabled in MetaPhlAn are:

• sensitive
• very-sensitive
• sensitive-local
• very-sensitive-local

[default very-sensitive]

--bowtie2_exe BOWTIE2_EXE

Full path and name of the BowTie2 executable. This option allows MetaPhlAn to reach the executable even when it is not in the system PATH or the system PATH is unreachable

--bowtie2out FILE_NAME

The file for saving the output of BowTie2

--no_map

Avoid storing the --bowtie2out map file

--tmp_dir

the folder used to store temporary files [default is the OS dependent tmp dir]

Post-mapping arguments

--tax_lev TAXONOMIC_LEVEL

The taxonomic level for the relative abundance output:
'a' : all taxonomic levels
'k' : kingdoms
'p' : phyla only
'c' : classes only
'o' : orders only
'f' : families only
'g' : genera only
's' : species only
[default 'a']

--min_cu_len

minimum total nucleotide length for the markers in a clade for estimating the abundance without considering sub-clade abundances [default 2000]

--min_alignment_len

The sam records for aligned reads with the longest subalignment length smaller than this threshold will be discarded. [default None]

--ignore_viruses

Do not profile viral organisms

--ignore_eukaryotes

Do not profile eukaryotic organisms

--ignore_bacteria

Do not profile bacterial organisms

--ignore_archaea

Do not profile archeal organisms

--stat_q

Quantile value for the robust average [default 0.1]

--ignore_markers IGNORE_MARKERS

File containing a list of markers to ignore.

--avoid_disqm

Deactivate the procedure of disambiguating the quasi-markers based on the marker abundance pattern found in the sample. It is generally recommended too keep the disambiguation procedure in order to minimize false positives

--stat

EXPERIMENTAL! Statistical approach for converting marker abundances into clade abundances
'avg_g' : clade global (i.e. normalizing all markers together) average
'avg_l' : average of length-normalized marker counts
'tavg_g' : truncated clade global average at --stat_q quantile
'tavg_l' : trunated average of length-normalized marker counts (at --stat_q)
'wavg_g' : winsorized clade global average (at --stat_q)
'wavg_l' : winsorized average of length-normalized marker counts (at --stat_q)
'med' : median of length-normalized marker counts
[default tavg_g]

Additional analysis types and arguments

-t ANALYSIS TYPE

Type of analysis to perform:

• rel_ab: profiling a metagenomes in terms of relative abundances
• rel_ab_w_read_stats: profiling a metagenomes in terms of relative abundances and estimate the number of reads coming from each clade.
• reads_map: mapping from reads to clades (only reads hitting a marker)
• clade_profiles: normalized marker counts for clades with at least a non-null marker
• marker_ab_table: normalized marker counts (only when > 0.0 and normalized by metagenome size if --nreads is specified)
• marker_counts: non-normalized marker counts [use with extreme caution]
• marker_pres_table: list of markers present in the sample (threshold at 1.0 if not differently specified with --pres_th

[default 'rel_ab']

--nreads NUMBER_OF_READS

The total number of reads in the original metagenome. It is used only when -t marker_table is specified for normalizing the length-normalized counts with the metagenome size as well. No normalization applied if --nreads is not specified

--pres_th PRESENCE_THRESHOLD

Threshold for calling a marker present by the -t marker_pres_table option

--clade

The clade for clade_specific_strain_tracker analysis

--min_ab

The minimum percentage abundace for the clade in the clade_specific_strain_tracker analysis

-h, --help

show this help message and exit

Output arguments

-o output file, --output_file output file

The output file (if not specified as positional argument)

--sample_id_key name

Specify the sample ID key for this analysis. Defaults to '#SampleID'.

--sample_id value

Specify the sample ID for this analysis. Defaults to 'Metaphlan2_Analysis'.

-s sam_output_file, --samout sam_output_file

The sam output file

--biom biom_output, --biom_output_file biom_output

If requesting biom file output: The name of the output file in biom format

--mdelim mdelim, --metadata_delimiter_char mdelim

Delimiter for bug metadata: - defaults to pipe. e.g. the pipe in k__Bacteria|p__Proteobacteria

Other arguments

--nproc N

The number of CPUs to use for parallelizing the mapping [default 1, i.e. no parallelism]

-v, --version

Prints the current MetaPhlAn version and exit