Find what NCBI RefSeq genomes match or are contained within your sequence data using Mash MinHash with a Mash sketch database of 54,925 NCBI RefSeq Genomes.
Easiest way to install refseq_masher and all its dependencies is with Conda through the BioConda channel:
conda install -c bioconda refseq_masher
Otherwise you can install refseq_masher from PyPI with pip install refseq_masher, but you would need to manually install Mash v2.0+.
Other than Python 3.5/3.6, the only external dependency of refseq_masher is Mash v2.0+.
- Pandas
- NumPy
- Click
- pytest for running tests
If you run refseq_masher without any arguments, you should see the following usage info:
Usage: refseq_masher [OPTIONS] COMMAND [ARGS]...
Find the closest matching NCBI RefSeq genomes or the genomes contained in
your contigs or reads.
Options:
--version Show the version and exit.
-v, --verbose Logging verbosity (-v for logging warnings; -vvv for logging
debug info)
-h, --help Show this message and exit.
Commands:
contains Find the NCBI RefSeq genomes contained in...
matches Find NCBI RefSeq genome matches for an input...
refseq_masher has 2 commands:
-
matchesfor finding the closest NCBI RefSeq genome matches to your input sequences -
containsfor finding what RefSeq genomes are contained within your input sequences- useful for finding what genomes may be contained within your metagenomic sample
Usage: refseq_masher matches [OPTIONS] INPUT...
Find NCBI RefSeq genome matches for an input genome fasta file
Input is expected to be one or more FASTA/FASTQ files or one or more
directories containing FASTA/FASTQ files. Files can be Gzipped.
Options:
--mash-bin TEXT Mash binary path (default="mash")
-o, --output PATH Output file path (default="-"/stdout)
--output-type [tab|csv] Output file type (tab|csv)
-n, --top-n-results INTEGER Output top N results sorted by distance in
ascending order (default=5)
-m, --min-kmer-threshold INTEGER
Mash sketch of reads: "Minimum copies of
each k-mer required to pass noise filter for
reads" (default=8)
-h, --help Show this message and exit.
With the FNA.GZ file for Salmonella enterica subsp. enterica serovar Enteritidis str. CHS44:
# download sequence file
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/329/025/GCF_000329025.1_ASM32902v1/GCF_000329025.1_ASM32902v1_genomic.fna.gz
# find RefSeq matches
refseq_masher -vv matches GCF_000329025.1_ASM32902v1_genomic.fna.gz
Log:
2018-01-29 11:02:13,786 INFO: Collected 1 FASTA inputs and 0 read sets [in ...refseq_masher/refseq_masher/utils.py:185]
2018-01-29 11:02:13,786 INFO: Creating Mash sketch file for ...refseq_masher/GCF_000329025.1_ASM32902v1_genomic.fna.gz [in ...refseq_masher/refseq_masher/mash/sketch.py:24]
2018-01-29 11:02:14,055 INFO: Created Mash sketch file at "/tmp/GCF_000329025.1_ASM32902v1_genomic.msh" [in ...refseq_masher/refseq_masher/mash/sketch.py:40]
2018-01-29 11:02:14,613 INFO: Ran Mash dist successfully (output length=11647035). Parsing Mash dist output [in ...refseq_masher/refseq_masher/mash/dist.py:64]
2018-01-29 11:02:15,320 INFO: Parsed Mash dist output into Pandas DataFrame with 54924 rows [in ...refseq_masher/refseq_masher/mash/dist.py:67]
2018-01-29 11:02:15,321 INFO: Deleting temporary sketch file "/tmp/GCF_000329025.1_ASM32902v1_genomic.msh" [in ...refseq_masher/refseq_masher/mash/dist.py:72]
2018-01-29 11:02:15,321 INFO: Sketch file "/tmp/GCF_000329025.1_ASM32902v1_genomic.msh" deleted! [in ...refseq_masher/refseq_masher/mash/dist.py:74]
2018-01-29 11:02:15,322 INFO: Ran Mash dist on all input. Merging NCBI taxonomic information into results output. [in ...refseq_masher/refseq_masher/cli.py:88]
2018-01-29 11:02:15,323 INFO: Fetching all taxonomy info for 5 unique NCBI Taxonomy UIDs [in ...refseq_masher/refseq_masher/taxonomy.py:35]
2018-01-29 11:02:15,325 INFO: Dropping columns with all NA values (ncol=32) [in ...refseq_masher/refseq_masher/taxonomy.py:38]
2018-01-29 11:02:15,327 INFO: Columns with all NA values dropped (ncol=11) [in ...refseq_masher/refseq_masher/taxonomy.py:40]
2018-01-29 11:02:15,327 INFO: Merging Mash results with relevant taxonomic information [in ...refseq_masher/refseq_masher/taxonomy.py:41]
2018-01-29 11:02:15,329 INFO: Merged Mash results with taxonomy info [in ...refseq_masher/refseq_masher/taxonomy.py:43]
2018-01-29 11:02:15,329 INFO: Merged taxonomic info into results output [in ...refseq_masher/refseq_masher/cli.py:90]
2018-01-29 11:02:15,329 INFO: Reordering output columns [in ...refseq_masher/refseq_masher/cli.py:91]
2018-01-29 11:02:15,331 INFO: Writing output to stdout [in ...refseq_masher/refseq_masher/writers.py:16]
Output:
Table output to standard output:
sample top_taxonomy_name distance pvalue matching full_taxonomy taxonomic_subspecies taxonomic_species taxonomic_genus taxonomic_family taxonomic_order taxonomic_class taxonomic_phylum taxonomic_superkingdom subspecies serovar plasmid bioproject biosample taxid assembly_accession match_id
GCF_000329025.1_ASM32902v1_genomic Salmonella enterica subsp. enterica serovar Enteritidis str. CHS44 0.0 0.0 400/400 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales; Enterobacteriaceae; Salmonella; enterica; subsp. enterica; serovar Enteritidis; str. CHS44 Salmonella enterica subsp. enterica Salmonella enterica Salmonella Enterobacteriaceae Enterobacterales Gammaproteobacteria Proteobacteria Bacteria enterica Enteritidis PRJNA185053 SAMN01041154 702979 NZ_ALFF ./rcn/refseq-NZ-702979-PRJNA185053-SAMN01041154-NZ_ALFF-.-Salmonella_enterica_subsp._enterica_serovar_Enteritidis_str._CHS44.fna
...
[truncated output]
The top match is Salmonella enterica subsp. enterica serovar Enteritidis str. CHS44 with a distance of 0.0 and 400/400 sketches matching, which is what we expected. There's other taxonomic information available in the results table that may be useful.
If you have a metagenomic sample or maybe a sample with some contamination, you may be interested in seeing what's in your sample. You can do this with refseq_masher contains <INPUT>.
Usage: refseq_masher contains [OPTIONS] INPUT...
Find the NCBI RefSeq genomes contained in your sequence files using Mash
Screen
Input is expected to be one or more FASTA/FASTQ files or one or more
directories containing FASTA/FASTQ files. Files can be Gzipped.
Options:
--mash-bin TEXT Mash binary path (default="mash")
-o, --output PATH Output file path (default="-"/stdout)
--output-type [tab|csv] Output file type (tab|csv)
-n, --top-n-results INTEGER Output top N results sorted by identity in
ascending order (default=0/all)
-i, --min-identity FLOAT Mash screen min identity to report
(default=0.9)
-v, --max-pvalue FLOAT Mash screen max p-value to report
(default=0.01)
-p, --parallelism INTEGER Mash screen parallelism; number of threads to
spawn (default=1)
-h, --help Show this message and exit.
For this example, we're going to see what RefSeq genomes are contained within sample SAMEA1877340 from BioProject PRJEB1775.
Description from BioProject PRJEB1775:
Design, Setting and Patients Forty-five samples were selected from a set of fecal specimens obtained from patients with diarrhea during the 2011 outbreak of STEC O104:H4 in Germany. Samples were chosen to represent STEC-positive patients with a range of clinical conditions and colony counts together with a small number of patients with other infections (Campylobacter jejnuni, Clostridium difficile and Salmonella enterica). Samples were subjected to high-throughput sequencing on the Illumina MiSeq and HiSeq 2500, followed by bioinformatics analysis.
We're going to download the FASTQ files for ERR260489:
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR260/ERR260489/ERR260489_1.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR260/ERR260489/ERR260489_2.fastq.gzWe're going to run refseq_masher against these FASTQ files:
refseq_masher -vv contains --top-n-results 50 -p 12 -o containment-ERR260489.tab ERR260489_1.fastq.gz ERR260489_2.fastq.gzLog:
2018-01-29 10:59:25,849 INFO: Grouped 2 fastqs into 1 groups [in ...refseq_masher/refseq_masher/utils.py:174]
2018-01-29 10:59:25,849 INFO: Collected 0 FASTA inputs and 1 read sets [in ...refseq_masher/refseq_masher/utils.py:185]
2018-01-29 10:59:25,849 INFO: Running Mash Screen with NCBI RefSeq sketch database against sample "ERR260489" with inputs: ['../ERR260489_1.fastq.gz', '../ERR260489_2.fastq.gz'] [in ...refseq_masher/refseq_masher/mash/screen.py:44]
Loading ...refseq_masher/refseq_masher/data/RefSeqSketches.msh...
4669418 distinct hashes.
Streaming from 2 inputs...
Estimated distinct k-mers in pool: 206836855
Summing shared...
Computing coverage medians...
Writing output...
2018-01-29 11:00:19,665 INFO: Ran Mash Screen on all input. Merging NCBI taxonomic information into results output. [in ...refseq_masher/refseq_masher/cli.py:134]
2018-01-29 11:00:19,666 INFO: Fetching all taxonomy info for 23 unique NCBI Taxonomy UIDs [in ...refseq_masher/refseq_masher/taxonomy.py:35]
2018-01-29 11:00:19,669 INFO: Dropping columns with all NA values (ncol=32) [in ...refseq_masher/refseq_masher/taxonomy.py:38]
2018-01-29 11:00:19,671 INFO: Columns with all NA values dropped (ncol=12) [in ...refseq_masher/refseq_masher/taxonomy.py:40]
2018-01-29 11:00:19,671 INFO: Merging Mash results with relevant taxonomic information [in ...refseq_masher/refseq_masher/taxonomy.py:41]
2018-01-29 11:00:19,674 INFO: Merged Mash results with taxonomy info [in ...refseq_masher/refseq_masher/taxonomy.py:43]
2018-01-29 11:00:19,674 INFO: Merged taxonomic information into results output [in ...refseq_masher/refseq_masher/cli.py:136]
2018-01-29 11:00:19,674 INFO: Reordering output columns [in ...refseq_masher/refseq_masher/cli.py:137]
2018-01-29 11:00:19,677 INFO: Wrote output to "containment-ERR260489.tab" [in ...refseq_masher/refseq_masher/writers.py:20]
Output:
sample top_taxonomy_name identity shared_hashes median_multiplicity pvalue full_taxonomy taxonomic_subspecies taxonomic_species taxonomic_genus taxonomic_family taxonomic_order taxonomic_class taxonomic_phylum taxonomic_superkingdom subspecies serovar plasmid bioproject biosample taxid assembly_accession match_id taxonomic_species group match_comment
ERR260489 Bacteroides fragilis 1.0 400/400 786 0.0 Bacteria; FCB group; Bacteroidetes/Chlorobi group; Bacteroidetes; Bacteroidia; Bacteroidales; Bacteroidaceae; Bacteroides; fragilis Bacteroides fragilis Bacteroides Bacteroidaceae Bacteroidales Bacteroidia Bacteroidetes Bacteria pLV22a 817 ./rcn/refseq-NG-817-.-.-.-pLV22a-Bacteroides_fragilis.fna
... [1 row] ...
ERR260489 Escherichia coli O104:H4 str. E92/11 1.0 400/400 48 0.0 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales; Enterobacteriaceae; Escherichia; coli; O104:H4; str. E92/11 Escherichia coli Escherichia Enterobacteriaceae Enterobacterales Gammaproteobacteria Proteobacteria Bacteria pE9211p3 1090927 NZ_AHAU ./rcn/refseq-NZ-1090927-.-.-NZ_AHAU-pE9211p3-Escherichia_coli_O104_H4_str._E92_11.fna
... [3 rows] ...
ERR260489 Kingella kingae KKC2005004457 1.0 400/400 5 0.0 Bacteria; Proteobacteria; Betaproteobacteria; Neisseriales; Neisseriaceae; Kingella; kingae; KKC2005004457 Kingella kingae Kingella Neisseriaceae Neisseriales Betaproteobacteria Proteobacteria Bacteria unnamed 1229911 ./rcn/refseq-NG-1229911-.-.-.-unnamed-Kingella_kingae_KKC2005004457.fna
ERR260489 Bacteroides cellulosilyticus WH2 0.9998440000000001 399/400 772 0.0 Bacteria; FCB group; Bacteroidetes/Chlorobi group; Bacteroidetes; Bacteroidia; Bacteroidales; Bacteroidaceae; Bacteroides; cellulosilyticus; WH2 Bacteroides cellulosilyticus Bacteroides Bacteroidaceae Bacteroidales Bacteroidia Bacteroidetes Bacteria pBWH2B 1268240 NZ_ATFI ./rcn/refseq-NZ-1268240-.-.-NZ_ATFI-pBWH2B-Bacteroides_cellulosilyticus_WH2.fna
... [1 row] ...
ERR260489 Klebsiella pneumoniae 0.9998440000000001 399/400 4 0.0 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales; Enterobacteriaceae; Klebsiella; pneumoniae Klebsiella pneumoniae Klebsiella Enterobacteriaceae Enterobacterales Gammaproteobacteria Proteobacteria Bacteria pMRC151 573 ./rcn/refseq-NG-573-.-.-.-pMRC151-Klebsiella_pneumoniae.fna
... [37 rows] ...
Some of the top genomes contained in this sample are sorted by identity and median multiplicity are:
- Bacteroides fragilis - fully contained (400/400) and high multiplicity (768)
- Escherichia coli O104:H4 - fully contained (400/400) and median multiplicity of 48
- Kingella kingae - fully contained (400/400) and median multiplicity of 5
- Klebsiella pneumoniae - 399/400 sketches contained with median multiplicity of 4
So with Mash we are able to find that the sample contained the expected genomic data (especially E. coli O104:H4).
Copyright Government of Canada 2017
Written by: National Microbiology Laboratory, Public Health Agency of Canada
Licensed under the Apache License, Version 2.0 (the "License"); you may not use this work except in compliance with the License. You may obtain a copy of the License at:
http://www.apache.org/licenses/LICENSE-2.0
Unless required by applicable law or agreed to in writing, software distributed under the License is distributed on an "AS IS" BASIS, WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied. See the License for the specific language governing permissions and limitations under the License.
Gary van Domselaar: [email protected]