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Sampling focal sequences¶

genome-sampler can be used for sampling focal sequences in addition to context sequences. In fact, all of the exact same sampling approaches can be applied in exactly the same way. The only additional file you’ll need to do this (relative to the files used in genome-sampler: step-by-step tutorial) is a focal sequence metadata file. This can mirror the format of the context sequence metadata file. For more details on the format of these files, see Metadata file format.

Running in parallel¶

genome-sampler can be run in parallel to speed it up. This is done in different ways depending on whether you’re running the steps individually or through Snakemake.

If you’re using Snakemake, you need only pass additional cores (via snakemake --cores N). The parallelizable steps will automatically use the provided resources.

If you’re running the steps individually you can pass the --p-n-threads option to several of the commands. For example, sample-diversity is the slowest step in the workflow. You can provide the --p-n-threads parameter to run it in parallel:

qiime genome-sampler sample-diversity \
 --i-context-seqs filtered-context-seqs.qza \
 --p-percent-id 0.9995 \
 --o-selection diversity-selection.qza \
 --p-n-threads N

When running this command, you should set N to be the number of available processors or cores on a single node of your system. For example, on a cluster that has nodes with 28 cores, you could run:

qiime genome-sampler sample-diversity \
 --i-context-seqs filtered-context-seqs.qza \
 --p-percent-id 0.9995 \
 --o-selection diversity-selection.qza \
 --p-n-threads 28

This would use all of the resources on a single node of that cluster.

Pre-computed alignment mask¶

We provide support for masking alignments with the pre-computed mask provided here. Like the Downstream alignment and phylogenetics presented here, this is experimental for working with viral genomes. Let us know how it works for you. It would be very useful to hear about how your final results (e.g., a phylogenetic tree) computed using this mask compares to one created with your own custom mask.

Removing sequences present in both focal and context sequence collections¶

As many researchers are rapidly submitting their sequence data to public repositories, sometimes your focal sequences may be present in context sequences that you obtain from a public repository. You can use QIIME 2 to remove those sequences from your context sequence collection.

First, visualize your focal sequence and context sequence collections with QIIME 2 to confirm that you know what QIIME 2 considers to be the sequence identifiers. You can do this by running the following two commands:

qiime feature-table tabulate-seqs \
 --i-data focal-seqs.qza \
 --o-visualization focal-seqs.qzv

qiime feature-table tabulate-seqs \
 --i-data context-seqs.qza \
 --o-visualization context-seqs.qzv

Then, load the two visualizations with QIIME 2 View. The values in the Feature ID column are the sequence ids. If the ids of the sequences that you want to remove are the same across your two data sets, you can follow the steps under Shared identifiers. If the identifiers you want to remove are not the same across your two data sets, or you’re not sure, follow the steps under Differing identifiers.

Shared identifiers¶

If your sequence identifiers are identical across your focal and context sequence collections, you can remove context sequences that are also focal sequences as follows:

qiime feature-table filter-seqs\
 --i-data context-seqs.qza \
 --m-metadata-file focal-metadata.tsv \
 --p-exclude-ids \
 --o-filtered-data filtered-context-seqs.qza

All of your downstream analysis steps should then use the resulting filtered-context-seqs.qza file.

Alternatively, if you want to remove these sequences from your focal sequence collection, you could do this as follows:

qiime feature-table filter-seqs\
 --i-data focal-seqs.qza \
 --m-metadata-file context-metadata.tsv \
 --p-exclude-ids \
 --o-filtered-data filtered-focal-seqs.qza

Differing identifiers¶

If your sequence identifiers differ across your focal and context sequences (or you’re not sure if they’re the same or if they differ), you can perform a metadata-based filtering of your sequences. The easiest way to approach this is to add a new column to your context sequence metadata file (context-metadata.tsv in the tutorial), with a column header like is-focal-sequence. You can do this with your favorite spreadsheet editor. For all of the sequences you would like to remove, add the value TRUE to this column. For all of the sequences you would like to retain, add the value FALSE to this column. Then, save the file and run the following command:

qiime feature-table filter-seqs \
 --i-data context-seqs.qza \
 --m-metadata-file context-metadata.tsv \
 --p-where "[is_focal_sequence]='FALSE'" \
 --o-filtered-data filtered-context-seqs.qza

This will create a new file, filtered-context-seqs.qza, which only contains the sequences that you indicated were not focal sequences in the context metadata. All of your downstream analysis steps should then use the resulting filtered-context-seqs.qza file.

Alternatively, if you want to remove sequences from your focal sequence collection instead of from your context sequence collection, you can edit your focal sequence metadata file, and then use that to filter in the same way that context sequences were filtered here.