Welcome back again RSG friends! The second newsletter of the German Regional Student Group of the iSCB Student Council is out and for this month we are going to talk about single-cell sequencing techniques and possible discoveries that have been made thanks to their application. We will also talk about their limitations and computational techniques that best suit for handling such big datasets. The newsletter is split in three parts: two parts are related to single-cell techniques and how can be used to connect different scales of complexity in the genome and one part is related to the usage of Deep Learning, a machine learning technique that recently has been applied to genomics to predict regulatory genomic regions based only on the DNA methylation values. Have fun and see you again in April! Yours:
RGS Germany (ISCB Student Council) – Tommaso Andreani, Yvonne Gladbach, Neetika Nath, Nikos Papadopoulos
Single-cell epigenomics: Recording the past and predicting the future
Single-cell sequencing is an increasingly popular platform that allows the integration of different scales of complexity at high resolutions providing advanced implications for diagnosing and disease progression. The integration of various epigenome components with genomic measurement allows studying cellular heterogeneity at different scales as well as the discovery of new layers of molecular connectivity between the genome and its functional output.
One of the most studied epigenetic modification is 5-methylcytosine at CpG islands: bisulphite treatment converts unmethylated cytosine to thymine leaving the methylated cytosines unmodified. In this way, it is possible to distinguish and quantify unmethylated from the methylated cytosines. After quantification, the integrative analysis of these values with different classes of genomic layers such as the one obtained from scATAC-Seq and scNOME-Seq can be modelled to reveal useful connections among the different scales of complexity. This because the latter allows the detection of chromatin modified regions as well as actively transcribed regions bounded by transcription factors.
With the biological complexity comes the computational challenge in hands and the sparse coverage of processed single-cell epigenome datasets requires careful consideration during downstream analyses. Three main points deserve careful attention depending on the question: (1) adjustment for differences in global methylation, (2) pooling cells with similar epigenetic profiles and (3) model-based approach to impute missing information by prediction. At the end, the multitude of new single cell sequencing techniques will allow new levels of understanding at the systems level and also will provide new opportunities to extract concepts that were hidden inside the data but that emerged from the computational analysis.
DeepCpG: accurate prediction of single-cell DNA methylation states using deep learning
Analyzing data from Single-cell sequencing technology such as for DNA methylation has created new challenges in computational biology. Current experimental protocols have already provided unprecedented insights into the regulation and dynamics of DNA methylation in single cells and have uncovered new links between epigenetic and transcriptional heterogeneity. However, these protocols produce incomplete coverage or CpG regions, since only small amounts of genomic DNA are present in each cell. In order to enable genome-wide analysis, the first critical step is the development of prediction methods for missing methylation states.
DeepCpG is such an algorithm, developed for single cells methylation values. It is based on deep neural networks, and leverages associations between DNA sequence patterns and methylation states, as well as relationships between neighboring CpG sites. It learns DNA sequence and methylation patterns from the data and uses them to uncover previously known and de novo sequence motifs associated with methylation changes / variability.
But how can missing methylation be modeled? DeepCpG consists of three modules: (1) a DNA module to extract features from the DNA sequence, (2) a CpG module to extract features from the CpG neighborhood of all cells and (3) a multi-task Joint module that integrates the evidence from both modules to predict the methylation state of target CpG sites for multiple cells.
The modules are used to implement a feed forward artificial neural network also named as convolutional network, a machine learning method previously applied in image and video recognition as well as natural language processing.
Single-cell transcriptional profiling of a multicellular organism
Multicellular organisms have evolved complex structures such as tissues and organs composed of multiple different cell types. Different cell types emerge from progenitor populations to fulfill specific physiological and developmental functions. Until now, the expression of marker genes has been used to define cell types. For example, in the hematopoietic lineage, membrane proteins were commonly used to characterize the different blood cell types and differentiation stages. Today, next generation sequencing approaches at the single cell level allow profiling of the transcriptomes of multiple single cells. Specifically developed computational methods can then be used to cluster them in small groups belonging to a particular phenotype/cell type with a specific function.
Cao et al. have developed a barcoding strategy that allowed them to expand single-cell RNA-sequencing to profile nearly 50000 cells from Caenorhabditis elegans at the L2 larval stage. Clustering analysis of the transcriptomes distributed the 762 somatic cells of the L2 larva in 27 different groups, corresponding to broader and more specific cell types. Groups of cells with little morphological and transcriptional heterogeneity, like muscle cells, formed rather large, unspecific groups. For other cell types, like neurons or intestinal cells, a lot of subtypes consisting of as few as one cell in every worm were identified. The computational analysis was typical for a scRNA-seq experiment: once the reads passed the quality control, were mapped to the reference genome. Afterwards the reads were converted to transcript counts, resulting to a matrix of counts per gene per cell. This matrix was then clustered with t-distributed stochastic neighbor embedding (t-SNE), a non-linear dimensionality reduction technique. Each cluster produced by t-SNE was manual assigned to a cell type following literature research. Among the different cell types identified, neurons and intestinal cells were the most characterized. Integrative analyses from modENCODE of Chromatin and Transcription Factor binding sites data identified possible regulatory programs of these cells.
In conclusion, single-cell sorting coupled with RNA sequencing is becoming a powerful technique to identify different classes of cell types in multicellular organisms. This will be helpful for the description and discovery of different cells of any tissue and organ. Additionally, it will help decipher developmental pathways and understand their regulation. Already, the transcriptomic profile of a cell is increasingly used as a proxy of cell type. We are still far from understanding possible mechanisms that dictate cellular differentiation only with the usage of the transcriptome, but at the moment we can enjoy this “single cell journey” to investigate the multitude of cells that colonize our body.