Neural networks for estimating microbe-metabolite co-occurence probabilities.
conda create -n rhapsody python=3.5 tensorflow numpy scipy pandas scikit-bio tqdm pip biom-format h5py -c conda-forge conda activate rhapsody pip install rhapsody
If you are getting errors, it is likely because you have garbage channels under your .condarc. Make sure to delete your .condarc -- you shouldn't need it.
To get started you can run a quick example as follows. This will learn microbe-metabolite vectors (mmvec) which can be used to estimate microbe-metabolite conditional probabilities that are accurate up to rank.
rhapsody mmvec \ --otu-file data/otus.biom \ --metabolite-file data/ms.biom \ --summary-dir summary \ --results-file cv-results.csv \ --ranks-file ranks.csv
While this is running, you can open up another session and run
tensorboard --logdir . for diagnosis, see FAQs below for more details.
If you investigate the summary folder, you will notice that there are a number of files deposited.
See the following url for a more complete tutorial with real datasets.
More information can found under
If you want to make this qiime2 compatible, install this in your qiime2 conda environment (see qiime2 installation instructions here) and run the following
pip install git+https://github.com/biocore/rhapsody.git qiime dev refresh-cache
This should allow your q2 environment to recognize rhapsody. Before we test the qiime2 plugin, run the following commands to import an example dataset
qiime tools import \ --input-path data/otus.biom \ --output-path otus.qza \ --type FeatureTable[Frequency] qiime tools import \ --input-path data/ms.biom \ --output-path ms.qza \ --type FeatureTable[Frequency]
Then you can run mmvec
qiime rhapsody mmvec \ --i-microbes otus.qza \ --i-metabolites ms.qza \ --output-dir results
In the results, there are two files, namely
results/conditionals.qza. The conditional biplot is a biplot representation the
conditional probability matrix so that you can visualize these microbe-metabolite interactions in an exploratory manner. This can be directly visualized in
Emperor as shown below. We also have the estimated conditional probability matrix given in
which an be unzip to yield a tab-delimited table via
unzip results/conditionals. Each row can be ranked,
so the top most occurring metabolites for a given microbe can be obtained by identifying the highest co-occurrence probabilities for each microbe.
It is worth your time to investigate the logs (labeled under
logdir**) that are deposited using Tensorboard.
The actual logfiles within this directory are labeled
events.out.tfevents.* : more discussion on this later.
Tensorboard can be run via
tensorboard --logdir .
You may need to tinker with the parameters to get readable tensorflow results, namely
A description of these two graphs is outlined in the FAQs below.
Then you can run the following to generate a emperor biplot.
qiime emperor biplot \ --i-biplot results/conditional_biplot.qza \ --m-sample-metadata-file data/metabolite-metadata.txt \ --m-feature-metadata-file data/microbe-metadata.txt \ --o-visualization emperor.qzv
Here, the samples are metabolites and the arrows are microbes. The distance between points is given by the Aitchison distance between metabolites (which is equivalent to Lovell's proportionality). The distance between arrow tips is the Aitchison distance between microbes.
More information behind the parameters can found under
qiime rhapsody --help
Q: Looks like there are two different commands, a standalone script and a qiime2 interface. Which one should I use?!?
A: It'll depend on how deep in the weeds you'll want to get. For most intents and purposes, the qiime2 interface will more practical for most analyses. There are 4 major reasons why the standalone scripts are more preferable to the qiime2 interface, namely
Customized acceleration : If you want to bring down your runtime from a few days to a few hours, you may need to compile Tensorflow to handle hardware specific instructions (i.e. GPUs / SIMD instructions). It probably is possible to enable GPU compatiability within a conda environment with some effort, but since conda packages binaries, SIMD instructions will not work out of the box.
Checkpoints : If you are not sure how long your analysis should run, the standalone script can allow you record checkpoints, which can allow you to recover your model parameters. This enables you to investigate your model while the model is training.
More model parameters : The standalone script will return the bias parameters learned for each dataset (i.e. microbe and metabolite abundances). These are stored under the summary directory (specified by
--summary) under the names
U.txtcorresponds to the learned simplical coordinates of the microbes and
V.txtcorresponds to the learned simplical coordinates for the metabolties.
Vbias.txtare bias parameters required to make the microbes and metabolite comparable.
More model diagnostics : In addition, it will record more cross validation statistics, which can enable you to investigate which microbes have the lowest cross validation error. There are two files that are generated if --ranks-file is specified (there are scenarios where you can blow memory if you compute all of the ranks, which is why this is an optional parameter).
model_results.csvcontains some cross validation results relevant to the global model fit. There are 6 statistics reported, namely
- TP, FP, FN, TN : True positive, False positive, False negatives and True negatives on holdout samples using
--top-kas a threshold to evaluate the top most abundant metabolites. This primarily tests to see how well the microbes can predict which metabolites are the most abundant for a given sample.
Precision, Recall : Precision and recall statistics estimated from the TP, FP, FN and TN computed.
F1 score: Harmonic average of precision and recall. See wikipedia for more details
meanRK: The average Spearman rank correlation across all microbes. This measures how well on average each microbe can predict the metabolite ranks.
otu_cv_results.csvprovides a more detailed breakdown of the cross-validation results for each microbe for each sample using the same statistics as discussed above.
- TP, FP, FN, TN : True positive, False positive, False negatives and True negatives on holdout samples using
Q : You mentioned that you can use GPUs. How can you do that??
A : This can be done by running
pip install tensorflow-gpu in your environment. See details here.
At the moment, these capabilities are only available for the standalone CLI due to complications of installation. See the
--arm-the-gpu option in the standalone interface.
Q : Neural networks scare me - don't they overfit the crap out of your data?
A : Here, we are using shallow neural networks (so only two layers). This falls under the same regime as PCA and SVD. But just as you can overfit PCA/SVD, you can also overfit mmvec. Which is why we have Tensorboard enabled for diagnostics. You can visualize the
cv_rmse to gauge if there is overfitting -- if your run is strictly decreasing, then that is a sign that you are probably not overfitting. But this is not necessarily indicative that you have reach the optimal -- you want to check to see if
logloss has reached a plateau as shown above.
Q : I'm confused, what is Tensorboard?
A : Tensorboard is a diagnostic tool that runs in a web browser. To open tensorboard, make sure you’re in the rhapsody environment and cd into the folder you are running the script above from. Then run:
tensorboard --logdir .
Returning line will look something like:
TensorBoard 1.9.0 at http://Lisas-MacBook-Pro-2.local:6006 (Press CTRL+C to quit)
Open the website (highlighted in red) in a browser. (Hint; if that doesn’t work try putting only the port number (here it is 6006), adding localhost, localhost:6006). Leave this tab alone. Now any rhapsody output directories that you add to the folder that tensorflow is running in will be added to the webpage.
FIRST graph in Tensorflow; 'Prediction accuracy'. Labelled
This is a graph of the prediction accuracy of the model; the model will try to guess the metabolite intensitiy values for the testing samples that were set aside in the script above, using only the microbe counts in the testing samples. Then it looks at the real values and sees how close it was.
The second graph is the
likelihood - if your
likelihood values are plateaued, that is a sign that you have converged and reached at a local minima.
The x-axis is the number of iterations (meaning times the model is training across the entire dataset). Every time you iterate across the training samples, you also run the test samples and the averaged results are being plotted on the y-axis.
The y-axis is the average number of counts off for each feature. The model is predicting the sequence counts for each feature in the samples that were set aside for testing. So in the graph above it means that, on average, the model is off by ~0.75 intensity units, which is low. However, this is ABSOLUTE error not relative error (unfortunately we don't know how to compute relative errors because of the sparsity in these datasets).
You can also compare multiple runs with different parameters to see which run performed the best. If you are doing this, be sure to look at the
training_column example make the testing samples consistent across runs.
Q : What's up with the
A : That is used for cross-validation if you have a specific reproducibility question that you are interested in answering. It can also make it easier to compare cross validation results across runs. If this is specified, only samples labeled "Train" under this column will be used for building the model and samples labeled "Test" will be used for cross validation. In other words the model will attempt to predict the microbe abundances for the "Test" samples. The resulting prediction accuracy is used to evaluate the generalizability of the model in order to determine if the model is overfitting or not. If this argument is not specified, then 10 random samples will be chosen for the test dataset. If you want to specify more random samples to allocate for cross-validation, the
num-random-test-examples argument can be specified.
Q : What sort of parameters should I focus on when picking a good model?
A : There are 3 different parameters to focus on,
--output_prior options specifies the width of the prior distribution of the coefficients, where the
--input_prior is typically specific to microbes and the
--output_prior is specific to metabolites.
For a prior of 1, this means 99% of entries in the embeddings (typically given in the
V.txt files are within -3 and +3 (log fold change). The higher differential-prior is, the more parameters can have bigger changes, so you want to keep this relatively small. If you see overfitting (accuracy and fit increasing over iterations in tensorboard) you may consider reducing the
--output_prior in order to reduce the parameter space.
Another parameter worth thinking about is
--latent_dim, which controls the number of dimensions used to approximate the conditional probability matrix. This also specifies the dimensions of the microbe/metabolite embeddings
V.txt. The more dimensions this has, the more accurate the embeddings can be -- but the higher the chance of overfitting there is. The rule of thumb to follow is in order to fit these models, you need at least 10 times as many samples as there are latent dimensions (this is following a similar rule of thumb for fitting straight lines). So if you have 100 samples, you should definitely not have a latent dimension of more than 10. Furthermore, you can still overfit certain microbes and metabolites. For example, you are fitting a model with those 100 samples and just 1 latent dimension, you can still easily overfit microbes and metabolites that appear in less than 10 samples -- so even fitting models with just 1 latent dimension will require some microbes and metabolites that appear in less than 10 samples to be filtered out.
Q : What does a good model fit look like??
A : Again the numbers vary greatly by dataset. But you want to see the both the
cv_rmse curves decaying, and plateau as close to zero as possible.
Q : How long should I expect this program to run?
A : Both
batch-size contribute to determining how long the algorithm will run, namely
Number of iterations =
epoch # multiplied by the ( Total # of microbial reads /
This also depends on if your program will converge. The
learning-rate specifies the resolution (smaller step size = smaller resolution, but may take longer to converge). You will need to consult with Tensorboard to make sure that your model fit is sane. See this paper for more details on gradient descent: https://arxiv.org/abs/1609.04747
If you are running this on a CPU, 16 cores, a run that reaches convergence should take about 1 day.
If you have a GPU - you maybe able to get this down to a few hours. However, some finetuning of the
batch_size parameter maybe required -- instead of having a small
batch_size < 100, you'll want to bump up the
batch_size to between 1000 and 10000 to fully leverage the speedups available on the GPU.