MicrobeCensus is a fast and easy to use pipeline for estimating the average genome size (AGS) of a microbial community from metagenomic data.

In short, AGS is estimated by aligning reads to a set of universal single-copy gene families present in nearly all cellular microbes (Bacteria, Archaea, Fungi). Because these genes occur once per genome, the average genome size of a microbial community is inversely proportional to the fraction of reads which hit these genes.

Once AGS is obtained, it becomes possible to obtain the total coverage of microbial genomes present in a sample (genome equivalents = total bp sequenced/AGS in bp), which can be useful for normalizing gene abundances.

• Python dependencies (installed via setup.py): Numpy, BioPython
• Supported platforms: Mac OSX, Unix/Linux; Windows not currently supported
• Python version 2 or 3

Clone the repo:
git clone https://github.com/snayfach/MicrobeCensus

Or, download the latest release from: https://github.com/snayfach/MicrobeCensus/releases

Unpack the project as necessary and navigate to the installation directory:
cd /path/to/MicrobeCensus

Run setup.py. This will install any dependencies:
python setup.py install or
sudo python setup.py install to install as a superuser

Alternatively, MicrobeCensus can be installed using pip (may not be latest version):
pip install MicrobeCensus or
sudo pip install MicrobeCensus to install as a superuser, or
pip install --user MicrobeCensus to install in your home directory

You can also install using conda (may not be latest version):
conda install -c bioconda microbecensus

Although this is not recommended, users may wish to run MicrobeCensus without running setup.py.

Both BioPython and Numpy will both need to be already installed. You should be able to enter the following command in the python interpreter without getting an error:
>>> import Bio.SeqIO
>>> import numpy

Next, add the MicrobeCensus module to your PYTHONPATH environmental variable:
export PYTHONPATH=$PYTHONPATH:/path/to/MicrobeCensus or
echo -e "\nexport PYTHONPATH=\$PYTHONPATH:/path/to/MicrobeCensus" >> ~/.bash_profile to avoid entering the command in the future

Finally, add the scripts directory to your PATH environmental variable:
export PATH=$PATH:/path/to/MicrobeCensus/scripts or
echo -e "\nexport PATH=\$PATH:/path/to/MicrobeCensus/scripts" >> ~/.bash_profile to avoid entering the command in the future

Now, you should be able to enter the command into your terminal without getting an error:
run_microbe_census.py -h

After installing MicrobeCensus, we recommend testing the software:
cd /path/to/MicrobeCensus/test
python test_microbe_census.py

MicrobeCensus can either be run as a command-line script or imported to python as a module.

run_microbe_census.py [-options] seqfiles outfile

Input/Output (required):

• SEQFILES
  path to input metagenome(s)
  for paired-end metagenomes use commas to specify each file (ex: read_1.fq.gz,read_2.fq.gz)
  can be FASTQ/FASTA
  can be gzip (.gz) or bzip (.bz2) compressed
• OUTFILE
  path to output file containing AGS estimate

Pipeline throughput (optional):

• -n NREADS
  number of reads to sample from seqfile and use for AGS estimation.
  to use all reads set to large number, like 100000000
  (default = 2000000)
• -t THREADS
  number of threads to use for database search (default= 1)
• -e
  quit after average genome size is obtained and do not estimate the number of genome equivalents in SEQFILES.
  useful in combination with -n for quick tests (default = False)

Quality control (optional):

• -l {50,60,70,80,90,100,110,120,130,140,150,175,200,225,250,300,350,400,450,500}
  all reads trimmed to this length; reads shorter than this are discarded
  (default = median read length)
• -q MIN_QUALITY
  minimum base-level PHRED quality score (default = -5)
• -m MEAN_QUALITY
  minimum read-level PHRED quality score (default = -5)
• -d
  filter duplicate reads (default = False)
• -u MAX_UNKNOWN
  max percent of unknown bases per read (default = 100)

Misc options:

• -h, --help: show this help message and exit
• -v: print program's progress to stdout (default = False)
• -V, --version: show program's version number and exit
• -r RAPSEARCH
  path to external RAPsearch2 v2.15 binary.
  useful if precompiled RAPsearch2 v2.15 binary included with MicrobeCensus does not work on your system

First, import the module:
>>> from microbe_census import microbe_census

Next, setup your options and arguments, formatted as a dictionary. The path to your metagenome is the only requirement (default values will be used for all other options):
>>> args = {'seqfiles':['MicrobeCensus/microbe_census/example/example.fq.gz']}

If you have paired-end libraries, separate them with a comma:
>>> args = {'seqfiles':['seqfile_1.fq.gz', 'seqfile_2.fq.gz']}

Alternatively, other options can be specified:

>>> args = {
  'seqfiles':['MicrobeCensus/microbe_census/example/example.fq.gz'],
  'nreads':100000,
  'read_length':100,
  'threads':1,
  'min_quality':10,
  'mean_quality':10,
  'filter_dups':False,
  'max_unknown':0,
  'verbose':True}

Finally, the entire pipeline can be run by passing your arguments to the run_pipeline function. MicrobeCensus returns the estimated AGS of your metagenome, along with a dictionary of used arguments: average_genome_size, args = microbe_census.run_pipeline(args)

For normalization, you can also estimate the number of genome equivalents in your metagenome:
count_bases = microbe_census.count_bases(args['seqfiles'])
genome_equivalents = count_bases/average_genome_size

• When in doubt, use default parameters! In most cases, MicrobeCensus tries to pick the best parameters for you.
• For more accurate estimates of AGS, use -n to increase the number of reads sampled. The default value of 2,000,000 should give good results, but more reads may result in slightly more accurate estimates, particularly when AGS is very large.
• Don't use quality filtering options (-q, -m, -d, -u) if you plan on using MicrobeCensus for normalization. In this case, MicrobeCensus should be directly run on the metagenome you used for estimating gene-family abundances.
• Use -v/--verbose to print program progress

MicrobeCensus writes several temporary files to disk. The location where temporary files are written are determined by the environmental variable TMPDIR. You can change this location as follows:
export TMPDIR=/new/location/for/temorary/files

Parameters
metagenome: path to your metagenome(s)
reads_sampled: the number of reads sampled from the metagenome to estimate AGS
trimmed_length: reads were trimmed to this length to estimate AGS
min_quality: minimum per-base quality
mean_quality: minimum average-base quality
filter_dups: filter exact duplicate reads
max_unknown: filter reads where the % of Ns is greater than this

Results
average_genome_size: the average genome size (in bp) of your input metagenome
total_bases: the total number of base-pairs of your input metagenome
genome_equivalents: the total coverage of microbial genomes in your input metagenome

The number of genome equivalents can be used to normalize count data obtained from metagenomes using the statistic RPKG (reads per kb per genome equivalent). This is similar to the commonly used statistic RPKM, but instead of dividing by the number of total mapped reads, we divide by the number of genome equivalents:

RPKG = (reads mapped to gene)/(gene length in kb)/(genome equivalents)

Use case: We have two metagenomic libraries, L1 and L2, and we use MicrobeCensus to estimate the number of genome equivilants in each:

GE_L1 = 40
GE_L2 = 20

Next, we map reads from each library to a reference database which contains a gene of interest G. G is 1000 bp long. We get 100 reads mapped to gene G from each library:

LENGTH_G = 1,000 bp
MAPPED_READS_G_L1 = 100
MAPPED_READS_G_L2 = 100

Finally, we quantify RPKG for gene G in each library:

RPKG for G in L1 = (100 mapped reads)/(1 kb)/(40 GE) = 2.5
RPKG for G in L2 = (100 mapped reads)/(1 kb)/(20 GE) = 5.0

• Run times are for a 150 bp library. Expect longer/shorter runtimes depending on read length.

We have included scripts and documentation for retraining MicrobeCensus, using user-supplied training genomes and gene families. Documentation and scripts can be found under: MicrobeCensus/training

If you use MicrobeCensus, please cite:

Nayfach, S. and Pollard, K.S. Average genome size estimation improves comparative metagenomics and sheds light on the functional ecology of the human microbiome. Genome biology 2015;16(1):51.