Genome Prediction Tutorial

Introduction

This tutorial explains the initial steps in the PICRUSt pipeline that looks after formatting input tree and trait tables, running ancestral state reconstruction, and extending predictions to all tips in the reference tree.

Most users will not need to conduct these steps and can skip directly to the Metagenome Prediction Tutorial.

Essential Files

All files required run this tutorial are in tutorials/picrust_starting_files.zip. Descriptions of these files are as follows:

  • Reference Tree (.nwk)
    • This is a phylogenetic tree constructed from a marker gene (typically 16S), that has tips representing both sequenced genomes and non-sequenced genomes.
    • We will use the greengenes tree gg_tree.nwk.
  • Marker gene copy number (tab-delimited .tab)
    • This is a simple tab-delimited table with two columns. The first column contains the genome identfiers column ids (typically IMG), and the second column contains the number of copies of the marker gene (16S). The first row must contain header ids for the columns.
    • We will use the file IMG_16S_counts.tab.
  • Functional trait copy number (tab-delimited .tab)
    • Same format as the marker gene copy number table above with genome identifiers being the first column, but contains an additional column for every functional trait with the column id representing that trait (e.g. K00001).
    • We will use KEGG KO functions IMG_ko_counts.tab.
  • Mapping file for tree tip ids to genome ids (tab-delimited .tab)
    • Simple two column tab delimited file with first column containing ids of tips in the tree that have genome sequences. Second column contains genome identifiers from marker gene and functional trait copy number files.
    • We will use the file GG_to_IMGv350.txt.
    • (Note: This file is optional if the tips representing genomes in the reference tree exactly match genome identifiers in the trait tables).

Formatting tree and trait tables

format_tree_and_trait_table.py does numerous formatting and checks to the reference tree and the trait tables. The following steps are done for both the marker gene copy number table and functional trait copy number table.

  1. All internal nodes in the reference tree are checked for problematic characters and unlabelled internal nodes are given labels.
  2. A pruned tree is created that contains only tips that have copy number predictions from sequenced genomes.
  3. Any traits in the trait table that are not in the reference tree are removed.

Format the 16S marker gene copy number table:

format_tree_and_trait_table.py -t GG_tree.nwk -i IMG_16S_counts.tab -m GG_to_IMGv350.txt -o format/16S/

Format the KO functional trait copy number table:

format_tree_and_trait_table.py -t GG_tree.nwk -i IMG_ko_counts.tab -m GG_to_IMGv350.txt -o format/KEGG/

Each of the above two commands creates 3 output files in the directory specified by the ‘-o’ option.

  1. reference_tree.newick
  2. pruned_tree.newick
  3. trait_table.tab

Note: that the reference_tree.newick files from the two commands will be identical.

Ancestral State Reconstruction

ancestral_state_reconstruction.py runs ancestral state reconstruction (ASR) to make predictions for each trait for every internal node in the pruned tree.

Various methods of ASR can be chosen including ace_pic, wagner, ace_ml, and ace_reml. The first two methods are very fast while the latter two ML methods require parallelization to be finished in a suitable timeframe.

Input is a formatted trait table and a pruned tree.

Output is a tab delimited file with first column containing internal node labels from the tree and other columns containing predicted ASR counts for each trait.

First, we will use the default PICRUSt ASR method ace_pic on the 16S trait table and the pruned tree:

ancestral_state_reconstruction.py -i format/16S/trait_table.tab -t format/16S/pruned_tree.newick -o asr/16S_asr_counts.tab

Second, run the same method on the formatted KO trait table (with the pruned tree):

ancestral_state_reconstruction.py -i format/KEGG/trait_table.tab -t format/KEGG/pruned_tree.newick -o asr/KEGG_asr_counts.tab

(Optional) Running other ASR methods

Other ASR methods can be run using the -m option:

ancestral_state_reconstruction.py -i format/16S/trait_table.tab -t format/16S/pruned_tree.newick -o asr/16S_asr_counts.tab -m wagner

ML based ASR methods (ace_reml and ace_ml) require much longer computational times. Parallelization is supported for multiple platforms (e.g. SGE, Torque, etc.).

For example use -p option to turn on parallelization, -j sge to specify a SGE based cluster, and -n 200 to group commands into 200 jobs:

ancestral_state_reconstruction.py -i format/KEGG/trait_table.tab -t format/KEGG/pruned_tree.newick -o asr/KEGG_asr_counts.tab -m ace_ml -p -j sge -n 200

Genome (trait) Prediction

predict_traits.py extends ASR predictions from internal nodes and known genomes to unsequenced tips in the marker tree.

Input is the formatted trait table, the formatted reference tree, and the ASR output file.

Output is a biom formatted file with ‘Observations’ (e.g rows) as tree tip ids (e.g. genomes/OTUs) and ‘Samples’ (e.g columns) as functional traits.

Make predictions for 16S:

predict_traits.py -i format/16S/trait_table.tab -t format/16S/reference_tree.newick -r asr/16S_asr_counts.tab -o predict_traits/16S_precalculated.biom

Make predictions for KOs:

predict_traits.py -i format/KEGG/trait_table.tab -t format/KEGG/reference_tree.newick -r asr/KEGG_asr_counts.tab -o predict_traits/ko_precalculated.biom

The 16S_precalculated.biom and ko_precalculated.biom can be used with the -c option of normalize_by_copy_number.py and predict_metagenomes.py described in the Metagenome Prediction Tutorial.

(Optional) Limiting predictions to those in OTU table

predict_traits.py can take a long time to run if making predictions for all tips in the green genes reference tree (400k tips). Therefore, you can limit the number of predictions to only those in your metagenome OTU table (the ones you care about) using the -l option.

Make predictions for KOs for a given OTU table using -l option:

predict_traits.py -i format/KEGG/trait_table.tab -t format/KEGG/reference_tree.newick -r asr/KEGG_asr_counts.tab -l your_otu_table.tsv -o predict_traits/ko_precalculated.biom