sag-mg-recruit

Read recruitment pipeline for comparison of sag abundance across metagenomes

Dependencies:

python 2.7

Flash

bwa

samtools

bedtools

To install with conda:

# create conda virtual env
conda create --name smr python=2.7

# activate environment
source activate smr

# install dependencies using conda
conda install -c bioconda flash bwa bedtools samtools pysam biopython
conda install matplotlib pandas numpy

# optional if you plan on using the checkM functionality:
conda install -c bioconda checkm

# download this repo
wget https://github.com/BigelowLab/sag-mg-recruit/archive/master.zip

# unarchive repo
unzip master.zip

# navigate into repo and run install script
cd sag-mg-recruit-master
python setup.py install

# to leave sag-mg-recruit environment
source deactivate smr

Whenever you want to run sag-mg-recruit, activate your environment:

source activate smr

For instructions on how to run type:

sag-mg-recruit --help

which will return:

$ sag-mg-recruit --help
Usage: sag-mg-recruit [OPTIONS] INPUT_MG_TABLE INPUT_SAG_TABLE

Options:
  --outdir TEXT          directory location to place output files
  --cores INTEGER        number of cores to run on  [default: 8]
  --mmd FLOAT            for join step: mismatch density  [default: 0.05]
  --mino INTEGER         for join step: minimum overlap  [default: 35]
  --maxo INTEGER         for join step: maximum overlap  [default: 150]
  --minlen INTEGER       for alignment and mg read count: minimum alignment
                         length to include; minimum read size to include
                         [default: 150]
  --pctid INTEGER        for alignment: minimum percent identity to keep
                         within overlapping region  [default: 95]
  --overlap INTEGER      for alignment: percent read that must overlap with
                         reference sequence to keep  [default: 0]
  --log TEXT             name of log file, else, log sent to standard out
  --concatenate BOOLEAN  include concatenated SAG in analysis  [default: True]
  --checkm BOOLEAN       should checkm be run on the SAGs?  [default: True]
  --keep_coverage        if you want to keep the genome coverage table (large)
  -h, --help             Show this message and exit.

Input details:

input_mg_table should be a csv file created using the mg_template.xlsx within the program directory. The columns are:

• name: desired name used to refer to each metagenome; any string without spaces, special characters or "."
• mg_f: path to forward reads for metagenome
• mg_r: path to reverse metagenomic reads (if there are none, put "None" in this column)
• wgs_technology: specify whether the library was sequenced with either illumina (enter "illumina") or 454-pyrosequencing (enter "pyro")
• join: designate whether you want the metagenome to be joined or not; either True or False

See mg_template.xlsx for example table. Input your own information and save as a .csv file.

intput_sag_table is a csv formatted table with the following columns:

• sag_name: any string with no spaces or '.'
• fasta_file: None if sag should be masked, otherwise path to input fasta to be processed
• gbk_file: path to SAG's annotated gbk file if mask = True
• mask: boolean indicating whether the SAG should have the 16/23S sequences masked (TRUE) or not (FALSE)

See sag_template.xlsx for example table. Input your own information in excel and save as a .csv file.

Example input script:

for help:

sag-mg-recruit -h

to run with 95% identity alignment, 40 cores, minimum read length of 100:

sag-mg-recruit --outdir <path to output dir> --cores 40 --minlen 100  --pctid 95 --log recruitment.log <input mg table> <input sag table>

Output:

The program will create a new output directory with three sub-directories: coverage, mgs and sags which will contain various output files created during the recruitment process.

The final output table will be located in the main output directory, called "summary_table_pctidXX_minlenXX_overlapXX.txt".

That table has one row per mg-sag pair with the following columns:

• sag: SAG name
• metagenome: metagenome name
• Percent_scaffolds_with_any_coverage: percent of SAG scaffolds with any coverage
• Percent_of_reference_bases_covered: percent of SAG bases covered by at least one read
• Average_coverage: mean coverage across the SAG
• total_reads_recruited: total number of metagenomic reads recruited to the SAG
• mg_wgs_technology: metagenome sequencing technology used, designated in the input_mg_table
• mg_read_count: total number of reads used in the processed metagenome
• sag_completeness: % sag completeness as determined by checkm
• sag_total_bp: number of bp in input SAG fasta file
• sag_size_mbp: SAG size in megabasepairs (mbp)
• reads_per_mbp: number of reads recruited per SAG mbp
• prop_mgreads_per_mbp: proprtion metagenomic reads recruited per SAG mbp

Changing alignment parameters

After you've run sag-mg-recruit, you can re-run the analysis with changed --pctid, --overlap, or --minlen parameters much more quickly than the original run by using the same input files, the same output directory but changing the --pctid, --overlap and/or --minlen parameters.

For example, say your first run was:

sag-mg-recruit --outdir /home/smroutput --cores 40 --minlen 150  --pctid 95 --overlap 95 --log recruitment.log mgtbl.txt sagtbl.txt

Re-run with different minlen and pctid parameters:

sag-mg-recruit --outdir /home/smroutput --cores 40 --minlen 100  --pctid 90 --overlap 100 --log recruitment.log mgtbl.txt sagtbl.txt

Note a quirk of one of the dependencies (FLASH) which is used for the join step: if there are metagenomic reads that need to be joined, make sure the output directory is specificied as a relative rather than absolute path.

Other Functions:

Other scripts can be found in smr/scripts/

extract_smr_reads.py: Extracts unaligned and aligned reads from smr run, and writes to the smr run's coverage directory in fastq.gz format. Like the main sag-mg-recruit software, this can be run multiple times with different parameters. Parameters are included in the output file name.

Usage: extract_smr_reads.py [OPTIONS] SMR_DIR

Options:
  --minlen INTEGER   for alignment and mg read count: minimum alignment length
                     to include; minimum read size to include  [default: 150]
  --pctid INTEGER    for alignment: minimum percent identity to keep within
                     overlapping region  [default: 95]
  --overlap INTEGER  for alignment: percent read that must overlap with
                     reference sequence to keep  [default: 0]
  --cores INTEGER    how many cores  [default: 10]
  -h, --help         Show this message and exit.

Example command: extract_smr_reads.py --minlen 150 --pctid 95 /smr/output/directory/