A sourmash tutorial¶
This tutorial should run without modification on Ubuntu 15.10 (“wily”); it was developed on AWS ami-05384865 (us-west region).
You’ll need about 30 GB of free disk space to download the database, and about 1-2 GB of RAM to search it. The tutorial should take about 20 minutes total to run.
Installing sourmash¶
To install sourmash, run:
sudo apt-get -y update && \
sudo apt-get install -y python3.5-dev python3.5-venv make \
libc6-dev g++ zlib1g-dev
this installs Python 3.5.
Now, create a local software install and populate it with Jupyter and other dependencies:
python3.5 -m venv ~/py3
. ~/py3/bin/activate
pip install -U pip
pip install -U Cython
pip install -U jupyter jupyter_client ipython pandas matplotlib scipy scikit-learn khmer
pip install -U https://github.com/dib-lab/sourmash/archive/master.zip
Generate a signature for Illumina reads¶
Download some reads and a reference genome:
mkdir ~/data
cd ~/data
wget https://s3.amazonaws.com/public.ged.msu.edu/ecoli_ref-5m.fastq.gz
wget https://s3.amazonaws.com/public.ged.msu.edu/ecoliMG1655.fa.gz
Compute a scaled MinHash signature from our reads:
mkdir ~/sourmash
cd ~/sourmash
sourmash compute --scaled 10000 ~/data/ecoli_ref*pe*.fq.gz -o ecoli-reads.sig -k 31
Compare reads to assemblies¶
Use case: how much of the read content is contained in the reference genome?
Build a signature for an E. coli genome:
sourmash compute --scaled 10000 -k 31 ~/data/ecoliMG1655.fa.gz -o ecoli-genome.sig
and now evaluate containment, that is, what fraction of the read content is contained in the genome:
sourmash search -k 31 ecoli-reads.sig ecoli-genome.sig --containment
and you should see:
# running sourmash subcommand: search
loaded query: /home/ubuntu/data/ecoli_ref-5m... (k=31, DNA)
loaded 1 signatures from ecoli-genome.sig
1 matches:
similarity match
---------- -----
46.6% /home/ubuntu/data/ecoliMG1655.fa.gz
Try the reverse - why is it bigger?
sourmash search -k 31 ecoli-genome.sig ecoli-reads.sig --containment
Make and search a database quickly.¶
Suppose that we have a collection of signatures (made with sourmash compute as above) and we want to search it with our newly assembled
genome (or the reads, even!). How would we do that?
Let’s grab a sample collection of 50 E. coli genomes and unpack it –
mkdir ecoli_many_sigs
cd ecoli_many_sigs
curl -O -L https://github.com/dib-lab/sourmash/raw/master/data/eschericia-sigs.tar.gz
tar xzf eschericia-sigs.tar.gz
rm eschericia-sigs.tar.gz
cd ../
This will produce 50 files named ecoli-N.sig in the ecoli_many_sigs –
ls ecoli_many_sigs
Let’s turn this into an easily-searchable database with sourmash index –
sourmash index -k 31 ecolidb ecoli_many_sigs/*.sig
and now we can search!
sourmash search ecoli-genome.sig ecolidb.sbt.json -n 20
You should see output like this:
select query k=31 automatically.
loaded query: /home/ubuntu/data/ecoliMG1655.... (k=31, DNA)
loaded 0 signatures and 1 databases total.
49 matches; showing first 20:
similarity match
---------- -----
75.4% NZ_JMGW01000001.1 Escherichia coli 1-176-05_S4_C2 e117605...
72.2% NZ_GG774190.1 Escherichia coli MS 196-1 Scfld2538, whole ...
71.4% NZ_JMGU01000001.1 Escherichia coli 2-011-08_S3_C2 e201108...
70.1% NZ_JHRU01000001.1 Escherichia coli strain 100854 100854_1...
69.0% NZ_JH659569.1 Escherichia coli M919 supercont2.1, whole g...
64.9% NZ_JNLZ01000001.1 Escherichia coli 3-105-05_S1_C1 e310505...
63.0% NZ_MOJK01000001.1 Escherichia coli strain 469 Cleandata-B...
62.9% NZ_MOGK01000001.1 Escherichia coli strain 676 BN4_676_1_(...
62.0% NZ_JHDG01000001.1 Escherichia coli 1-176-05_S3_C1 e117605...
59.9% NZ_MIWF01000001.1 Escherichia coli strain AF7759-1 contig...
52.7% NZ_KE700241.1 Escherichia coli HVH 147 (4-5893887) acYxy-...
51.7% NZ_APWY01000001.1 Escherichia coli 178200 gec178200.conti...
49.3% NZ_LVOV01000001.1 Escherichia coli strain swine72 swine72...
49.3% NZ_MIWP01000001.1 Escherichia coli strain K6412 contig_00...
49.0% NZ_LQWB01000001.1 Escherichia coli strain GN03624 GCID_EC...
48.9% NZ_JHGJ01000001.1 Escherichia coli O45:H2 str. 2009C-4780...
48.1% NZ_CP011331.1 Escherichia coli O104:H4 str. C227-11, comp...
47.7% NZ_JHNB01000001.1 Escherichia coli O103:H25 str. 2010C-45...
47.7% NZ_JHRE01000001.1 Escherichia coli strain 302014 302014_1...
47.6% NZ_JHHE01000001.1 Escherichia coli O103:H2 str. 2009C-327...
Compare many signatures and build a tree.¶
Compare all the things:
sourmash compare ecoli_many_sigs/* -o ecoli_cmp
and then plot:
sourmash plot --pdf --labels ecoli_cmp
which will produce a file ecoli_cmp.matrix.pdf and ecoli_cmp.dendro.pdf
which you can then download via your file browser and view on your local
computer.
Here’s a PNG version:
What’s in my metagenome?¶
Download and unpack a newer version of the k=31 RefSeq index described in CTB’s blog post – this one contains sketches of all 100k Genbank microbes. (See available databases for more information.)
curl -O https://s3-us-west-1.amazonaws.com/spacegraphcats.ucdavis.edu/microbe-genbank-sbt-k31-2017.05.09.tar.gz
tar xzf microbe-genbank-sbt-k31-2017.05.09.tar.gz
This produces a file genbank-k31.sbt.json and a whole bunch of hidden
files in the directory .sbt.genbank-k31.
Next, run the ‘gather’ command to see what’s in your ecoli genome –
sourmash gather -k 31 ecoli-genome.sig genbank-k31.sbt.json
and you should get:
# running sourmash subcommand: gather
loaded query: /home/ubuntu/data/ecoliMG1655.... (k=31, DNA)
overlap p_query p_match
--------- ------- --------
4.9 Mbp 100.0% 99.8% CP011320.1 Escherichia coli strain SQ...
found 1 matches total;
the recovered matches hit 100.0% of the query
In this case, the output is kind of boring because this is a single genome. But! You can use this on metagenomes (assembled and unassembled) as well; you’ve just got to make the signature files.
To see this in action, here is gather running on a signature generated from some sequences that assemble (but don’t align to known genomes) from the Shakya et al. 2013 mock metagenome paper.
wget https://github.com/dib-lab/sourmash/raw/master/doc/_static/shakya-unaligned-contigs.sig
sourmash gather -k 31 shakya-unaligned-contigs.sig genbank-k31.sbt.json
This should yield:
loaded query: mqc500.QC.AMBIGUOUS.99.unalign... (k=31, DNA)
loaded 0 signatures and 1 databases total.
overlap p_query p_match
--------- ------- --------
1.4 Mbp 11.0% 58.0% JANA01000001.1 Fusobacterium sp. OBRC...
1.0 Mbp 7.7% 25.9% CP001957.1 Haloferax volcanii DS2 pla...
0.9 Mbp 7.4% 11.8% BA000019.2 Nostoc sp. PCC 7120 DNA, c...
0.7 Mbp 5.9% 23.0% FOVK01000036.1 Proteiniclasticum rumi...
0.7 Mbp 5.3% 17.6% AE017285.1 Desulfovibrio vulgaris sub...
0.6 Mbp 4.9% 11.1% CP001252.1 Shewanella baltica OS223, ...
0.6 Mbp 4.8% 27.3% AP008226.1 Thermus thermophilus HB8 g...
0.6 Mbp 4.4% 11.2% CP000031.2 Ruegeria pomeroyi DSS-3, c...
480.0 kbp 3.8% 7.6% CP000875.1 Herpetosiphon aurantiacus ...
410.0 kbp 3.3% 10.5% CH959317.1 Sulfitobacter sp. NAS-14.1...
1.4 Mbp 2.2% 11.8% LN831027.1 Fusobacterium nucleatum su...
0.5 Mbp 2.1% 5.3% CP000753.1 Shewanella baltica OS185, ...
420.0 kbp 1.9% 7.7% FNDZ01000023.1 Proteiniclasticum rumi...
150.0 kbp 1.2% 4.5% CP015081.1 Deinococcus radiodurans R1...
150.0 kbp 1.2% 8.2% CP000969.1 Thermotoga sp. RQ2, comple...
290.0 kbp 1.1% 4.1% CH959311.1 Sulfitobacter sp. EE-36 sc...
1.2 Mbp 1.0% 5.0% CP013328.1 Fusobacterium nucleatum su...
110.0 kbp 0.9% 3.5% FREL01000833.1 Enterococcus faecalis ...
0.6 Mbp 0.8% 2.8% CP000527.1 Desulfovibrio vulgaris DP4...
340.0 kbp 0.6% 3.3% KQ235732.1 Fusobacterium nucleatum su...
70.0 kbp 0.6% 1.2% CP000850.1 Salinispora arenicola CNS-...
60.0 kbp 0.5% 0.7% CP000270.1 Burkholderia xenovorans LB...
50.0 kbp 0.4% 2.6% CP001080.1 Sulfurihydrogenibium sp. Y...
50.0 kbp 0.4% 3.2% L77117.1 Methanocaldococcus jannaschi...
found less than 40.0 kbp in common. => exiting
found 24 matches total;
the recovered matches hit 73.1% of the query
If you use the -o flag, gather will write out a csv that contains additional information. The column headers and their meanings are:
- intersect_bp: the approximate number of base pairs in common between the query and the match
- f_orig_query: fraction of original query; the fraction of the original query that is contained within the match
- f_match: fraction of match; the fraction of the match that is contained within the query
- f_unique_to_query: fraction unique to query; the fraction of the query that uniquely overlaps with the match
- f_unique_weighted: fraction unique to query weighted by abundance; fraction unique to query, weighted by abundance in the query
It is straightforward to build your own databases for use with search
and gather; ping us if you want us to write that up.