This page deals with common issues in running the analysis. For issues with installing or running the software please raise an issue on github.

Known bugs

When I look at my clusters on a tree, they make no sense

This is a bug caused by alphabetic sorting of labels in PopPUNK >=v2.0.0 with pp-sketchlib <v1.5.1. There are three ways to fix this:

  • Upgrade to PopPUNK >=v2.2 and pp-sketchlib >=v1.5.1 (preferred).
  • Run scripts/ on your .dists.pkl file and re-run model fits.
  • Create the database with poppunk_sketch --sketch and poppunk_sketch --query directly, rather than PopPUNK --create-db.

I have updated PopPUNK, and my clusters still seemed scrambled

This is possible using query assignment with --update-db, or in some cases with --gpu-dists. Please update to PopPUNK >=v2.4.0 with pp-sketchlib >=v1.7.0

Calculating distances using 0 thread(s)

This will lead to an error later on in execution. This is due to a version mismatch between PopPUNK and pp-sketchlib. Installation of both packages via conda should keep the versions compatible, but there are ways they can get out of sync.

The solution is as above: upgrade to PopPUNK >=v2.2 and pp-sketchlib >=v1.5.1.

Error/warning messages

Row name mismatch

PopPUNK may throw:

RuntimeError: Row name mismatch. Old: 6999_2#17.fa,6259_5#6.fa
New: 6952_7#16.fa,6259_5#6.fa

This is an error where the mash output order does not match the order in stored databases (.pkl). Most likely, the input files are from different runs, possibly due to using --overwrite. Run again, giving each step its own output directory.

Samples are missing from the final network

When running --assign-query an error such as:

WARNING: Samples 7553_5#54.fa,6999_5#1.fa are missing from the final network

Means that samples present in --distances and or --ref-db are not present in the loaded network. This should be considered an error as it will likely lead to other errors and warnings. Make sure the provided network is the one created by applying the --model-dir to --distances, and that the same output directory has not been used and overwriten by different steps or inputs.

Old cluster split across multiple new clusters

When running --assign-query, after distances have been calculated and queries are being assigned warnings such as:

WARNING: Old cluster 1 split across multiple new clusters

Mean that a single cluster in the original clustering is now split into more than one cluster. This means something has gone wrong, as the addition of new queries should only be able to merge existing clusters, not cause them to split.

Most likely, the --previous-clustering directory is inconsistent with the --ref-db and/or --model-dir. Make sure the clusters are those created from the network being used to assign new queries.

If you want to change cluster names or assign queries to your own cluster definitions you can use the --external-clustering argument instead.

Memory/run-time issues

Here are some tips based on experiences analysing larger datasets:

  • Add --threads – they are used fairly efficiently throughout.
  • Consider the --gpu-sketch and --gpu-dists options is applicable, and a GPU is available.
  • In --refine-model set --pos-shift 0 to avoid creating huge networks with close to \(N^2\) edges. Mixture models normally need to be pruned.
  • In --refine-model you may add the --no-local option to skip that step and decrease run-time, though gains are likely marginal.
  • Use --rapid-nj, if producing microreact output.

Another option for scaling is to run --create-db with a smaller initial set (not using the --full-db command), then use --assign-query to add to this.