Protein function prediction is a difficult bioinformatics problem. Many recent methods use deep neural networks to learn complex sequence representations and predict function from these. Deep supervised models require a lot of labeled training data which are not available for this task. However, a very large amount of protein sequences without functional labels is available. We applied an existing deep sequence model that had been pretrained in an unsupervised setting on the supervised task of protein molecular function prediction. We found that this complex feature representation is effective for this task, outperforming hand-crafted features such as one-hot encoding of amino acids, k-mer counts, secondary structure and backbone angles. Also, it partly negates the need for complex prediction models, as a two-layer perceptron was enough to achieve competitive performance in the third Critical Assessment of Functional Annotation benchmark. We also show that combining this sequence representation with protein 3D structure information does not lead to performance improvement, hinting that 3D structure is also potentially learned during the unsupervised pretraining. Implementations of all used models can be found at https://github.com/stamakro/GCN-for-Structure-and-Function. Supplementary data are available at Bioinformatics online.

The current rate at which new DNA and protein sequences are being generated is too fast to experimentally discover the functions of those sequences, emphasizing the need for accurate Automatic Function Prediction (AFP) methods. AFP has been an active and growing research field for decades and has made considerable progress in that time. However, it is certainly not solved. In this paper, we describe challenges that the AFP field still has to overcome in the future to increase its applicability. The challenges we consider are how to: (1) include condition-specific functional annotation, (2) predict functions for non-model species, (3) include new informative data sources, (4) deal with the biases of Gene Ontology (GO) annotations, and (5) maximally exploit the GO to obtain performance gains. We also provide recommendations for addressing those challenges, by adapting (1) the way we represent proteins and genes, (2) the way we represent gene functions, and (3) the algorithms that perform the prediction from gene to function. Together, we show that AFP is still a vibrant research area that can benefit from continuing advances in machine learning with which AFP in the 2020s can again take a large step forward reinforcing the power of computational biology.

Most automatic functional annotation methods assign Gene Ontology (GO) terms to proteins based on annotations of highly similar proteins. We advocate that proteins that are less similar are still informative. Also, despite their simplicity and structure, GO terms seem to be hard for computers to learn, in particular the Biological Process ontology, which has the most terms (>29 000). We propose to use Label-Space Dimensionality Reduction (LSDR) techniques to exploit the redundancy of GO terms and transform them into a more compact latent representation that is easier to predict. We compare proteins using a sequence similarity profile (SSP) to a set of annotated training proteins. We introduce two new LSDR methods, one based on the structure of the GO, and one based on semantic similarity of terms. We show that these LSDR methods, as well as three existing ones, improve the Critical Assessment of Functional Annotation performance of several function prediction algorithms. Cross-validation experiments on Arabidopsis thaliana proteins pinpoint the superiority of our GO-aware LSDR over generic LSDR. Our experiments on A.thaliana proteins show that the SSP representation in combination with a kNN classifier outperforms state-of-the-art and baseline methods in terms of cross-validated F-measure. Source code for the experiments is available at https://github.com/stamakro/SSP-LSDR. Supplementary data are available at Bioinformatics online.

Cell-free DNA (cfDNA) are DNA fragments originating from dying cells that are detectable in bodily fluids, such as the plasma. Accelerated cell death, for example caused by disease, induces an elevated concentration of cfDNA. As a result, determining the cell type origins of cfDNA molecules can provide information about an individual’s health. In this work, we aim to increase the sensitivity of methylation-based cell type deconvolution by adapting an existing method, CelFiE, which uses the methylation beta values of individual CpG sites to estimate cell type proportions. Our new method, CelFEER, instead differentiates cell types by the average methylation values within individual reads. We additionally improved the originally reported performance of CelFiE by using a new approach for finding marker regions that are differentially methylated between cell types. We show that CelFEER estimates cell type proportions with a higher correlation (r = 0.94 ± 0.04) than CelFiE (r = 0.86 ± 0.09) on simulated mixtures of cell types. Moreover, we show that the cell type proportion estimated by CelFEER can differentiate between ALS patients and healthy controls, between pregnant women in their first and third trimester, and between pregnant women with and without gestational diabetes.

Co-expression of two genes across different conditions is indicative of their involvement in the same biological process. However, when using RNA-Seq datasets with many experimental conditions from diverse sources, only a subset of the experimental conditions is expected to be relevant for finding genes related to a particular Gene Ontology (GO) term. Therefore, we hypothesize that when the purpose is to find similarly functioning genes, the co-expression of genes should not be determined on all samples but only on those samples informative for the GO term of interest. To address this, we developed Metric Learning for Co-expression (MLC), a fast algorithm that assigns a GO-term-specific weight to each expression sample. The goal is to obtain a weighted co-expression measure that is more suitable than the unweighted Pearson correlation for applying Guilt-By-Association-based function predictions. More specifically, if two genes are annotated with a given GO term, MLC tries to maximize their weighted co-expression and, in addition, if one of them is not annotated with that term, the weighted co-expression is minimized. Our experiments on publicly available Arabidopsis thaliana RNA-Seq data demonstrate that MLC outperforms standard Pearson correlation in term-centric performance. Moreover, our method is particularly good at more specific terms, which are the most interesting. Finally, by observing the sample weights for a particular GO term, one can identify which experiments are important for learning that term and potentially identify novel conditions that are relevant, as demonstrated by experiments in both A. thaliana and Pseudomonas Aeruginosa. MLC is available as a Python package at www.github.com/stamakro/MLC. Supplementary data are available at Bioinformatics online.

Deep generative models, such as variational autoencoders (VAE), have gained increasing attention in computational biology due to their ability to capture complex data manifolds which subsequently can be used to achieve better performance in downstream tasks, such as cancer type prediction or subtyping of cancer. However, these models are difficult to train due to the large number of hyperparameters that need to be tuned. To get a better understanding of the importance of the different hyperparameters, we examined six different VAE models when trained on TCGA transcriptomics data and evaluated on the downstream tasks of cluster agreement with cancer subtypes and survival analysis. We studied the effect of the latent space dimensionality, learning rate, optimizer, initialization and activation function on the quality of subsequent downstream tasks on the TCGA samples. We foundβ-TCVAE and DIP-VAE to have a good performance, on average, despite being more sensitive to hyperparameters selection. Based on these experiments, we derived recommendations for selecting the different hyperparameters settings. To ensure generalization, we tested all hyperparameter configurations on the GTEx dataset. We found a significant correlation (ρ= 0.7) between the hyperparameter effects on clustering performance in the TCGA and GTEx datasets. This highlights the robustness and generalizability of our recommendations. In addition, we examined whether the learned latent spaces capture biologically relevant information. Hereto, we measured the correlation and mutual information of the different representations with various data characteristics such as gender, age, days to metastasis, immune infiltration, and mutation signatures. We found that for all models the latent factors, in general, do not uniquely correlate with one of the data characteristics nor capture separable information in the latent factors even for models specifically designed for disentanglement.