# Tutorial¶

This tutorial will guide you through the use of the four modes of PopPUNK, explaining when to use each one. In places we refer to Troubleshooting which explains how to deal with common problems when the defaults don’t quite work.

The first two steps can be run together in a single command using --easy-run, which in many cases will work without need for further modification.

In this tutorial we will work with the Salmonella genomes reviewed by Alikhan et al which can be downloaded from EnteroBase.

## Creating a database¶

To analyse a population from scratch (where PopPUNK hasn’t been used before) the first step is to create a PopPUNK database, which is essentially a list of all the core and accessory distances between each pair of isolates in the collection.

The basic command to do this is as follows:

poppunk --create-db --r-files reference_list.txt --output strain_db --threads 2 --plot-fit 5


Where references.txt is a list of fasta assemblies to analyse, created by, for example:

ls assemblies/*.fasta > reference_list.txt


The references will first be hashed at different k-mer lengths, then pairwise distances are calculated, which are finally converted into core and accessory distances:

PopPUNK (POPulation Partitioning Using Nucleotide Kmers)
Mode: Building new database from input sequences
Creating mash database for k = 13
Random 13-mer probability: 0.06
Creating mash database for k = 17
Random 17-mer probability: 0.00
Creating mash database for k = 15
Random 15-mer probability: 0.00
Creating mash database for k = 19
Random 19-mer probability: 0.00
Creating mash database for k = 21
Random 21-mer probability: 0.00
Creating mash database for k = 25
Random 25-mer probability: 0.00
Creating mash database for k = 23
Random 23-mer probability: 0.00
Creating mash database for k = 27
Random 27-mer probability: 0.00
Creating mash database for k = 29
Random 29-mer probability: 0.00
mash dist -p 2 ./strain_db/strain_db.13.msh ./strain_db/strain_db.13.msh 2> strain_db.err.log
mash dist -p 2 ./strain_db/strain_db.15.msh ./strain_db/strain_db.15.msh 2> strain_db.err.log
mash dist -p 2 ./strain_db/strain_db.17.msh ./strain_db/strain_db.17.msh 2> strain_db.err.log
mash dist -p 2 ./strain_db/strain_db.19.msh ./strain_db/strain_db.19.msh 2> strain_db.err.log
mash dist -p 2 ./strain_db/strain_db.21.msh ./strain_db/strain_db.21.msh 2> strain_db.err.log
mash dist -p 2 ./strain_db/strain_db.23.msh ./strain_db/strain_db.23.msh 2> strain_db.err.log
mash dist -p 2 ./strain_db/strain_db.25.msh ./strain_db/strain_db.25.msh 2> strain_db.err.log
mash dist -p 2 ./strain_db/strain_db.27.msh ./strain_db/strain_db.27.msh 2> strain_db.err.log
mash dist -p 2 ./strain_db/strain_db.29.msh ./strain_db/strain_db.29.msh 2> strain_db.err.log
Calculating core and accessory distances

Done


We would recommend using as many threads as available for maximum speed (even if #threads > #k-mers). To convert k-mer distances into core and accessory distances the following relationship is used:

$\begin{split}& \mathrm{pr}(a, b) = (1-a)(1-c)^k \\ & \log (\mathrm{pr}(a, b)) = \log(1-a) + k \cdot \log(1-c)\end{split}$

Where $$\mathrm{pr}(a, b)$$ is the proportion of k-mers matching at length $$k$$ between sequences $$a$$ and $$b$$. In log-linear space this is linear by k-mer length, and a constrained least squared fit gives the accessory distance (the intercept) and the core distance (the slope).

Warning

A straight line fit is required for correct calculation of core and accessory distances. To inspect this the use of the --plot-fit options is generally recommended to inspect some of the regressions. Choice of min-k depends on this, and is discussed in Choosing the right k-mer lengths.

### Output files¶

This will create two files strain_db/strain_db.dists.npy and strain_db/strain_db.dists.pkl which store the distances and strain names respectively. These are then used in Fitting the model.

There are also databases of sketches at each k-mer length (*.msh) which can be re-used if the same data is fitted with a new range of k-mer lengths. Otherwise they should be recalculated by specifying --overwrite.

### Relevant command line options¶

The following command line options can be used in this mode:

Mode of operation:
 --create-db Create pairwise distances database between reference sequences
Input files:
 --r-files R_FILES File listing reference input assemblies
Output options:
 --output OUTPUT Prefix for output files (required) --plot-fit PLOT_FIT Create this many plots of some fits relating k-mer to core/accessory distances [default = 0] --overwrite Overwrite any existing database files
Quality control options:
 --max-a-dist MAX_A_DIST Maximum accessory distance to permit [default = 0.5] --ignore-length Ignore outliers in terms of assembly length [default = False]
Kmer comparison options:
 --min-k MIN_K Minimum kmer length [default = 9] --max-k MAX_K Maximum kmer length [default = 29] --k-step K_STEP K-mer step size [default = 4] --sketch-size SKETCH_SIZE Kmer sketch size [default = 10000]
Other options:
 --mash MASH Location of mash executable --threads THREADS Number of threads to use during database querying [default = 1] --no-stream Use temporary files for mash dist interfacing. Reduce memory use/increase disk use for large datasets

## Fitting the model¶

The basic command used to fit the model is as follows:

poppunk-runner.py --fit-model --distances strain_db/strain_db.dists --output strain_db --full-db --ref-db strain_db --K 2


This will fit a mixture of up to three 2D Gaussians to the distribution of core and accessory distances:

PopPUNK (POPulation Partitioning Using Nucleotide Kmers)
Mode: Fitting model to reference database

Fit summary:
Avg. entropy of assignment        0.0042
Number of components used 2
Network summary:
Components        12
Density   0.1852
Transitivity      0.9941
Score     0.8100

Done


The default is to fit two components, one for between-strain and one for within-strain distances. There are a number of summary statistics which you can use to assess the fit:

Statistic Interpretation
Avg. entropy of assignment How confidently each distance is assigned to a component. Closer to zero is more confident, and indicates less overlap of components, which may be indicative of less recombination overall.
Number of components used The number of mixture components actually used, which may be less than the maximum allowed.
Components The number of components in the network == the number of population clusters
Density The proportion of edges in the network. 0 is no links, 1 is every link. Lower is better.
Transitivity The transitivity of the network, between 0 and 1. Higher is better
Score Network score based on density and transitivity. Higher is better.

Important

This is the most important part of getting a good estimation of population structure. In many cases choosing a sensible --K will get a fit with a good score, but in more complex cases PopPUNK allows alternative model fitting. See Refining a model for a discussion on how to improve the model fit.

The most useful plot is strain_db_DPGMM_fit.png which shows the clustering:

This looks reasonable. The component closest to the origin is used to create a network where isolates determined to be within the same strain are linked by edges. The connected components of this network are then the population clusters.

In this case, allowing more components (--K 10) gives a worse fit as more complexity is introduced arbitrarily:

PopPUNK (POPulation Partitioning Using Nucleotide Kmers)
Mode: Fitting model to reference database

Fit summary:
Avg. entropy of assignment      0.0053
Number of components used       10
Network summary:
Components      121
Density 0.0534
Transitivity    0.8541
Score   0.8085

Done


In this case the fit is too conservative, and the network has a high number of components.

Once you have a good fit, run again with the --microreact option (and --rapidnj if you have rapidnj installed). This will create output files which can dragged and dropped into Microreact for visualisation of the results.

Drag the files strain_db_microreact_clusters.csv, strain_db_perplexity5.0_accessory_tsne, and strain_db_core_NJ_microreact.nwk onto Microreact. For this example, the output is at https://microreact.org/project/Skg0j9sjz (this also includes a CSV of additional metadata downloaded from EnteroBase and supplied to PopPUNK with --info-csv).

The left panel shows the tree from the core distances, and the right panel the embedding of accessory distances (at perplexity 30). Differences in clustering between the two can be informative of separate core and accessory evolution, but in this case they are correlated as expected for strains. Tips are coloured by the PopPUNK inferred cluster.

Note

t-SNE can be sensitive to the --perplexity parameter provided. This can be re-run as necessary by changing the parameter value. Use a value between 5 and 50, but see Setting the perplexity parameter for t-SNE for further discussion.

### Using DBSCAN¶

Clustering can also be performed by using DBSCAN, which uses the HDBSCAN* library. Run the same fit-model command as above, but add the --dbscan option:

poppunk-runner.py --fit-model --distances strain_db/strain_db.dists --output strain_db --full-db --ref-db strain_db --dbscan


The output is as follows:

PopPUNK (POPulation Partitioning Using Nucleotide Kmers)
Mode: Fitting model to reference database

Fit summary:
Number of clusters      5
Number of datapoints    100000
Number of assignments   100000
Network summary:
Components      9
Density 0.1906
Transitivity    0.9979
Score   0.8077

Done


In this case the fit is quite similar to the mixture model:

The small black points are classified as noise, and are not used in the network construction.

Warning

If there are a lot of noise points (in black) then fit refinement will be subsequently required, as these points will not contribute to the network. See Refining a model.

### Use of full-db¶

By default the --full-db option is off. When on this will keep every sample in the analysis in the database for future querying.

When off (the default) representative samples will be picked from each cluster by choosing only one reference sample from each clique (where all samples in a clqiue have a within-cluster link to all other samples in the clique). This can significantly reduce the database size for future querying without loss of accuracy. Representative samples are written out to a .refs file, and a new database is sketched for future distance comparison.

In the case of the example above, this reduces from 848 to 14 representatives (one for each of the twelve clusters, except for 3 and 6 which have two each).

If the program was run through using --full-db, references can be picked and a full directory with a PopPUNK model for query assignment created using the poppunk_references program:

poppunk_references --network strain_db/strain_db_graph.gpickle --ref-db strain_db --distances strain_db/strain_db.dists \
--model strain_db --output strain_references --threads 4


Using the --model will also copy over the model fit, so that the entire PopPUNK database is in a single directory.

### Providing previous cluster definitions¶

By using the option --external-clustering one can provide cluster names or labels that have been previously defined by any other method. This could include, for example, another clustering methods IDs, serotypes, clonal complexes and MLST assignments. The input is a CSV file which is formatted as follows:

sample,serotype,MLST
sample1,12,34
sample2,23F,1


This can contain any subset of the samples in the input, and additionally defined samples will be safely ignored.

PopPUNK will output a file _external_clusters.csv which has, for each sample in the input (either reference or query, depending on the mode it was run in), a list of of these labels which have been assigned to any sample in the PopPUNK cluster. Samples are expected to have a single label, but may receive multiple labels. Novel query clusters will not receive labels. An example of output:

sample,serotype,MLST
sample1,12,34
sample2,23F,1
sample3,15B;15C,21
sample4,NA,NA


### Output files¶

• strain_db.search.out – the core and accessory distances between all pairs.
• strain_db_graph.gpickle – the network used to predict clusters.
• strain_db_DPGMM_fit.png – scatter plot of all distances, and mixture model fit and assignment.
• strain_db_DPGMM_fit_contours.png – contours of likelihood function fitted to data (blue low -> yellow high). The thick red line is the decision boundary between within- and between-strain components.
• strain_db_distanceDistribution.png – scatter plot of the distance distribution fitted by the model, and a kernel-density estimate.
• strain_db.csv – isolate names and the cluster assigned.
• strain_db.png – unclustered distribution of distances used in the fit (subsampled from total).
• strain_db.npz – save fit parameters.
• strain_db.refs – representative references in the new database (unless --full-db was used).

If --dbscan was used:

• strain_db_dbscan.png – scatter plot of all distances, and DBSCAN assignment.

If --external-clustering was used:

• strain_db_external_clusters.csv – a CSV file relating the samples to previous clusters provided in the input CSV.

If --microreact was used:

• strain_db_core_dists.csv – matrix of pairwise core distances.
• strain_db_acc_dists.csv – matrix of pairwise accessory distances.
• strain_db_core_NJ_microreact.nwk – neighbour joining tree using core distances (for microreact).
• strain_db_perplexity5.0_accessory_tsne.dot – t-SNE embedding of accessory distances at given perplexity (for microreact).
• strain_db_microreact_clusters.csv – cluster assignments plus any epi data added with the --info-csv option (for microreact).

If --cytoscape was used:

• strain_db_cytoscape.csv – cluster assignments plus any epi data added with the --info-csv option (for cytoscape).
• strain_db_cytoscape.graphml – XML representation of resulting network (for cytoscape).

### Relevant command line options¶

The following command line options can be used in this mode:

Mode of operation:
 --fit-model Fit a mixture model to a reference database
Input files:
 --ref-db REF_DB Location of built reference database --distances DISTANCES Prefix of input pickle of pre-calculated distances --external-clustering EXTERNAL_CLUSTERING File with cluster definitions or other labels generated with any other method.
Output options:
 --output OUTPUT Prefix for output files (required) --full-db Keep full reference database, not just representatives --overwrite Overwrite any existing database files
Quality control options:
 --max-a-dist MAX_A_DIST Maximum accessory distance to permit [default = 0.5]
Model fit options:
 --K K Maximum number of mixture components [default = 2] --dbscan Use DBSCAN rather than mixture model --D D Maximum number of clusters in DBSCAN fitting [default = 100] --min-cluster-prop MIN_CLUSTER_PROP Minimum proportion of points in a cluster in DBSCAN fitting [default = 0.0001]
Further analysis options:
 --microreact Generate output files for microreact visualisation --cytoscape Generate network output files for Cytoscape --phandango Generate phylogeny and TSV for Phandango visualisation --grapetree Generate phylogeny and CSV for grapetree visualisation --rapidnj RAPIDNJ Path to rapidNJ binary to build NJ tree for Microreact --perplexity PERPLEXITY Perplexity used to calculate t-SNE projection (with –microreact) [default=20.0] --info-csv INFO_CSV Epidemiological information CSV formatted for microreact (can be used with other outputs)
Other options:
 --mash MASH Location of mash executable

Note

If using the default mixture model threads will only be used if --full-db is not specified and sketching of the representatives is performed at the end.

## Refining a model¶

In species with a relatively high recombination rate the distinction between the within- and between-strain distributions may be blurred in core and accessory space. This does not give the mixture model enough information to draw a good boundary as the likelihood is very flat in this region.

See this example of 616 S. pneumoniae genomes with the DPGMM fit. These genomes were collected from Massachusetts, first reported here and can be accessed here.

Although the score of this fit looks ok (0.904), inspection of the network and microreact reveals that it is too liberal and clusters have been merged. This is because some of the blur between the origin and the central distribution has been included, and connected clusters together erroneously.

The likelihood of the model fit and the decision boundary looks like this:

Using the core and accessory distributions alone does not give much information about exactly where to put the boundary, and the only way to fix this would be by specifying strong priors on the weights of the distributions. Fortunately the network properties give information in the region, and we can use --refine-fit to tweak the existing fit and pick a better boundary.

Run:

poppunk --refine-model --distances strain_db/strain_db.dists --output strain_db --full-db --ref-db strain_db --threads 4


Briefly:

• A line between the within- and between-strain means is constructed
• The point on this line where samples go from being assigned as within-strain to between-strain is used as the starting point
• A line normal to the first line, passing through this point is constructed. The triangle formed by this line and the x- and y-axes is now the decision boundary. Points within this line are within-strain.
• The starting point is shifted by a distance along the first line, and a new decision boundary formed in the same way. The network is reconstructed.
• The shift of the starting point is optimised, as judged by the network score. First globally by a grid search, then locally near the global optimum.

If the mixture model does not give any sort of reasonable fit to the points, see Using fit refinement when mixture model totally fails for details about how to set the starting parameters for this mode manually.

The score is a function of transitivity (which is expected to be high, as everything within a cluster should be the same strain as everything else in the cluster) and density (which should be low, as there are far fewer within- than between-strain links).

Here is the refined fit, which has a score of 0.939, and 62 rather than 32 components:

Which, looking at the microreact output, is much better:

The core and accessory distances can also be used on their own. Add the --indiv-refine option to refine the fit to these two distances independently (see Using core/accessory only for more information).

### Output files¶

The files are as for --fit-model (Output files), and also include:

• strain_db_refined_fit.png – A plot of the new linear boundary, and core and accessory distances coloured by assignment to either side of this boundary.
• strain_db_refined_fit.npz – The saved parameters of the refined fit.

If --indiv-refine was used, a copy of the _clusters.csv and network .gpickle files for core and accessory only will also be produced.

### Relevant command line options¶

The following command line options can be used in this mode:

Mode of operation:
 --refine-model Refine the accuracy of a fitted model
Input files:
 --ref-db REF_DB Location of built reference database --distances DISTANCES Prefix of input pickle of pre-calculated distances --external-clustering EXTERNAL_CLUSTERING File with cluster definitions or other labels generated with any other method.
Output options:
 --output OUTPUT Prefix for output files (required) --full-db Keep full reference database, not just representatives --overwrite Overwrite any existing database files
Quality control options:
 --max-a-dist MAX_A_DIST Maximum accessory distance to permit [default = 0.5]
Refine model options:
 --pos-shift POS_SHIFT Maximum amount to move the boundary away from origin [default = 0.2] --neg-shift NEG_SHIFT Maximum amount to move the boundary towards the origin [default = 0.4] --manual-start MANUAL_START A file containing information for a start point. See documentation for help. --indiv-refine Also run refinement for core and accessory individually --no-local Do not perform the local optimization step (speed up on very large datasets)
Further analysis options:
 --microreact Generate output files for microreact visualisation --cytoscape Generate network output files for Cytoscape --phandango Generate phylogeny and TSV for Phandango visualisation --grapetree Generate phylogeny and CSV for grapetree visualisation --rapidnj RAPIDNJ Path to rapidNJ binary to build NJ tree for Microreact --perplexity PERPLEXITY Perplexity used to calculate t-SNE projection (with –microreact) [default=20.0] --info-csv INFO_CSV Epidemiological information CSV formatted for microreact (can be used with other outputs)
Other options:
 --mash MASH Location of mash executable --threads THREADS Number of threads to use during database querying [default = 1]

Note

Threads are used for the global optimisation step only. If the local optimisation step is slow, turn it off with --no-local.

## Assigning queries¶

Once a database has been built and a model fitted (either in one step with --easy-run, or having run both steps separately) new sequences can be assigned to a cluster using --assign-queries. This process is much quicker than building a database of all sequences from scratch, and will use the same model fit as before. Cluster names will not change, unless queries cause clusters to be merged (in which case they will be the previous cluster names, underscore separated).

Having created a file listing the new sequences to assign query_list.txt, the command to assign a cluster to new sequences is:

poppunk --assign-query --ref-db strain_db --q-files query_list.txt --output strain_query --threads 3 --update-db


Where strain_db is the output of the previous PopPUNK commands, containing the model fit and distances.

Note

It is possible to specify a model fit in a separate directory from the distance sketches using --model-dir. Similarly a clustering and network can be specified using --previous-clustering.

First, distances between queries and sequences in the reference database will be calculated. The model fit (whether mixture model, DBSCAN or refined) will be loaded and used to determine matches to existing clusters:

PopPUNK (POPulation Partitioning Using Nucleotide Kmers)
Mode: Assigning clusters of query sequences

Creating mash database for k = 15
Random 15-mer probability: 0.00
Creating mash database for k = 13
Random 13-mer probability: 0.04
Creating mash database for k = 17
Random 17-mer probability: 0.00
Creating mash database for k = 19
Random 19-mer probability: 0.00
Creating mash database for k = 21
Random 21-mer probability: 0.00
Creating mash database for k = 23
Random 23-mer probability: 0.00
Creating mash database for k = 25
Random 25-mer probability: 0.00
Creating mash database for k = 27
Random 27-mer probability: 0.00
Creating mash database for k = 29
Random 29-mer probability: 0.00
mash dist -p 3 ./strain_db/strain_db.13.msh ./strain_query/strain_query.13.msh 2> strain_db.err.log
mash dist -p 3 ./strain_db/strain_db.15.msh ./strain_query/strain_query.15.msh 2> strain_db.err.log
mash dist -p 3 ./strain_db/strain_db.17.msh ./strain_query/strain_query.17.msh 2> strain_db.err.log
mash dist -p 3 ./strain_db/strain_db.19.msh ./strain_query/strain_query.19.msh 2> strain_db.err.log
mash dist -p 3 ./strain_db/strain_db.21.msh ./strain_query/strain_query.21.msh 2> strain_db.err.log
mash dist -p 3 ./strain_db/strain_db.23.msh ./strain_query/strain_query.23.msh 2> strain_db.err.log
mash dist -p 3 ./strain_db/strain_db.25.msh ./strain_query/strain_query.25.msh 2> strain_db.err.log
mash dist -p 3 ./strain_db/strain_db.27.msh ./strain_query/strain_query.27.msh 2> strain_db.err.log
mash dist -p 3 ./strain_db/strain_db.29.msh ./strain_query/strain_query.29.msh 2> strain_db.err.log
Calculating core and accessory distances


If query sequences were found which didn’t match an existing cluster they will start a new cluster. PopPUNK will check whether any of these novel clusters should be merged, based on the model fit:

Found novel query clusters. Calculating distances between them:
Creating mash database for k = 13
Random 13-mer probability: 0.04
Creating mash database for k = 15
Random 15-mer probability: 0.00
Creating mash database for k = 17
Random 17-mer probability: 0.00
Creating mash database for k = 19
Random 19-mer probability: 0.00
Creating mash database for k = 21
Random 21-mer probability: 0.00
Creating mash database for k = 23
Random 23-mer probability: 0.00
Creating mash database for k = 25
Random 25-mer probability: 0.00
Creating mash database for k = 27
Random 27-mer probability: 0.00
Creating mash database for k = 29
Random 29-mer probability: 0.00
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.13.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.13.msh 2> ./strain_dbij_sqnjr_tmp.err.log
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.15.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.15.msh 2> ./strain_dbij_sqnjr_tmp.err.log
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.17.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.17.msh 2> ./strain_dbij_sqnjr_tmp.err.log
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.19.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.19.msh 2> ./strain_dbij_sqnjr_tmp.err.log
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.21.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.21.msh 2> ./strain_dbij_sqnjr_tmp.err.log
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.23.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.23.msh 2> ./strain_dbij_sqnjr_tmp.err.log
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.25.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.25.msh 2> ./strain_dbij_sqnjr_tmp.err.log
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.27.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.27.msh 2> ./strain_dbij_sqnjr_tmp.err.log
mash dist -p 3 ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.29.msh ././strain_dbij_sqnjr_tmp/./strain_dbij_sqnjr_tmp.29.msh 2> ./strain_dbij_sqnjr_tmp.err.log
Calculating core and accessory distances


At this point, cluster assignments for the query sequences are written to a CSV file. Finally, if new clusters were created due to the queries, the database will be updated to reflect this if --update-db was used:

Creating mash database for k = 13
Random 13-mer probability: 0.04
Overwriting db: ./strain_query/strain_query.13.msh
Creating mash database for k = 15
Random 15-mer probability: 0.00
Overwriting db: ./strain_query/strain_query.15.msh
Creating mash database for k = 17
Random 17-mer probability: 0.00
Overwriting db: ./strain_query/strain_query.17.msh
Creating mash database for k = 19
Random 19-mer probability: 0.00
Overwriting db: ./strain_query/strain_query.19.msh
Creating mash database for k = 21
Random 21-mer probability: 0.00
Overwriting db: ./strain_query/strain_query.21.msh
Creating mash database for k = 23
Random 23-mer probability: 0.00
Overwriting db: ./strain_query/strain_query.23.msh
Creating mash database for k = 25
Random 25-mer probability: 0.00
Overwriting db: ./strain_query/strain_query.25.msh
Creating mash database for k = 27
Random 27-mer probability: 0.00
Overwriting db: ./strain_query/strain_query.27.msh
Creating mash database for k = 29
Random 29-mer probability: 0.00
Overwriting db: ./strain_query/strain_query.29.msh
Writing strain_query/strain_query.13.joined.msh...
Writing strain_query/strain_query.15.joined.msh...
Writing strain_query/strain_query.17.joined.msh...
Writing strain_query/strain_query.19.joined.msh...
Writing strain_query/strain_query.21.joined.msh...
Writing strain_query/strain_query.23.joined.msh...
Writing strain_query/strain_query.25.joined.msh...
Writing strain_query/strain_query.27.joined.msh...
Writing strain_query/strain_query.29.joined.msh...

Done


Note

For future uses of --assign-query, the database now stored in strain-query should be used as the --ref-db argument.

### Using core/accessory only¶

In some cases, such as analysis within a lineage, it may be desirable to use only core or accessory distances to classify further queries. This can be achieved by using the --core-only or --accessory-only options with a fit produced by Refining a model. The default is to use the x-axis intercept of the boundary as the core distance cutoff (y-axis for accessory). However, if planning on using this mode we recommend running the refinement with the --indiv-refine options, which will allow these boundaries to be placed independently, allowing the best fit in each case:

poppunk --refine-model --distances strain_db/strain_db.dists --output strain_db --full-db --indiv-refine --ref-db strain_db --threads 4
PopPUNK (POPulation Partitioning Using Nucleotide Kmers)
Mode: Refining model fit using network properties

Initial boundary based network construction
Decision boundary starts at (0.54,0.36)
Trying to optimise score globally
Trying to optimise score locally
Refining core and accessory separately
Initial boundary based network construction
Decision boundary starts at (0.54,0.36)
Trying to optimise score globally
Trying to optimise score locally
Initial boundary based network construction
Decision boundary starts at (0.54,0.36)
Trying to optimise score globally
Trying to optimise score locally
Network summary:
Components      132
Density 0.0889
Transitivity    0.9717
Score   0.8853
Network summary:
Components      114
Density 0.0955
Transitivity    0.9770
Score   0.8837
Network summary:
Components      92
Density 0.0937
Transitivity    0.9327
Score   0.8453
writing microreact output:
Building phylogeny
Running t-SNE

Done


There are three different networks, and the core and accessory boundaries will also be shown on the refined_fit.png plot as dashed gray lines:

### Output files¶

The main output is strain_query/strain_query_clusters.csv, which contains the cluster assignments of the query sequences, ordered by frequency.

If --update-db was used a full updated database will be written to --output, which consists of sketches at each k-mer length (*.msh), a search.out file of distances, and a .gpickle of the network.

### Relevant command line options¶

The following command line options can be used in this mode:

Mode of operation:
 --assign-query Assign the cluster of query sequences without re- running the whole mixture model
Input files:
 --ref-db REF_DB Location of built reference database --q-files Q_FILES File listing query input assemblies --external-clustering EXTERNAL_CLUSTERING File with cluster definitions or other labels generated with any other method.
Output options:
 --output OUTPUT Prefix for output files (required) --update-db Update reference database with query sequences
Quality control options:
 --max-a-dist MAX_A_DIST Maximum accessory distance to permit [default = 0.5] --ignore-length Ignore outliers in terms of assembly length [default = False]
Database querying options:
 --model-dir MODEL_DIR Directory containing model to use for assigning queries to clusters [default = reference database directory] --previous-clustering PREVIOUS_CLUSTERING Directory containing previous cluster definitions and network [default = use that in the directory containing the model] --core-only Use a core-distance only model for assigning queries [default = False] --accessory-only Use an accessory-distance only model for assigning queries [default = False]
Other options:
 --mash MASH Location of mash executable --threads THREADS Number of threads to use [default = 1] --no-stream Use temporary files for mash dist interfacing. Reduce memory use/increase disk use for large datasets --version show program’s version number and exit

## Creating external visualisations from a fitted model¶

Visualisations for external software (Microreact etc) will be created in a mode calling --fit-model, --refine-model or --assign-query if any of the following options were added:

• --microreact
• --cytoscape
• --phandango
• --grapetree

Additionally, if --refine-model, --indiv-refine and --cytoscape are all specified, the networks for core and accessory distances only will also be output.

To create these outputs for visualisation after the initial command has been run use the --generate-viz mode, with the same options as the original run (plus any specific to the visualisation). In this mode you may also specify a file containing a list of samples to include in the visualisation with --subset.

Note

Only a single network will be used in this mode if core and accessory distance restricted models have also been produced. To visualise these instead of the combined fit use --core-only or --accessory-only.

## Using a previous model with a new database¶

If you have a model which has been fitted which you wish to apply this to a new reference database, you may do this with --use-model. This will take a fitted model, apply it to distances from --create-db and produce a network, assignment and reference database for future use with --assign-query.

Note

Generally, to use an existing model with new data it is better to --assign-query (see Assigning queries). This mode can be used when the model, reference database and network are out of sync due to accidentally overwriting one or losing track of versions.

Options are the same as --fit-model for GMM and DBSCAN models or --refine-model for refined models.