Automated Segmentation of Cells with IHC Membrane Staining

This study presents a fully automated membrane segmentation technique for immunohistochemical tissue images with membrane staining, which is a critical task in computerized immunohistochemistry (IHC). Membrane segmentation is particularly tricky in immunohistochemical tissue images because the cellular membranes are visible only in the stained tracts of the cell, while the unstained tracts are not visible. Our automated method provides accurate segmentation of the cellular membranes in the stained tracts and reconstructs the approximate location of the unstained tracts using nuclear membranes as a spatial reference. Accurate cell-by-cell membrane segmentation allows per cell morphological analysis and quantification of the target membrane proteins that is fundamental in several medical applications such as cancer characterization and classification, personalized therapy design, and for any other applications requiring cell morphology characterization. Experimental results on real datasets from different anatomical locations demonstrate the wide applicability and high accuracy of our approach in the context of IHC analysi

FuGePrior: A novel gene fusion prioritization algorithm based on accurate fusion structure analysis in cancer RNA-seq samples

Abstract Background Latest Next Generation Sequencing technologies opened the way to a novel era of genomic studies, allowing to gain novel insights into multifactorial pathologies as cancer. In particular gene fusion detection and comprehension have been deeply enhanced by these methods. However, state of the art algorithms for gene fusion identification are still challenging. Indeed, they identify huge amounts of poorly overlapping candidates and all the reported fusions should be considered for in lab validation clearly overwhelming wet lab capabilities. Results In this work we propose a novel methodological approach and tool named FuGePrior for the prioritization of gene fusions from paired-end RNA-Seq data. The proposed pipeline combines state of the art tools for chimeric transcript discovery and prioritization, a series of filtering and processing steps designed by considering modern literature on gene fusions and an analysis on functional reliability of gene fusion structure. Conclusions FuGePrior performance has been assessed on two publicly available paired-end RNA-Seq datasets: The first by Edgren and colleagues includes four breast cancer cell lines and a normal breast sample, whereas the second by Ren and colleagues comprises fourteen primary prostate cancer samples and their paired normal counterparts. FuGePrior results accounted for a reduction in the number of fusions output of chimeric transcript discovery tools that ranges from 65 to 75% depending on the considered breast cancer cell line and from 37 to 65% according to the prostate cancer sample under examination. Furthermore, since both datasets come with a partial validation we were able to assess the performance of FuGePrior in correctly prioritizing real gene fusions. Specifically, 25 out of 26 validated fusions in breast cancer dataset have been correctly labelled as reliable and biologically significant. Similarly, 2 out of 5 validated fusions in prostate dataset have been recognized as priority by FuGePrior tool

Optimizing Splicing Junction Detection in Next Generation Sequencing Data on a Virtual-GRID Infrastructure

The new protocol for sequencing the messenger RNA in a cell, named RNA-seq produce millions of short sequence fragments. Next Generation Sequencing technology allows more accurate analysis but increase needs in term of computational resources. This paper describes the optimization of a RNA-seq analysis pipeline devoted to splicing variants detection, aimed at reducing computation time and providing a multi-user/multisample environment. This work brings two main contributions. First, we optimized a well-known algorithm called TopHat by parallelizing some sequential mapping steps. Second, we designed and implemented a hybrid virtual GRID infrastructure allowing to efficiently execute multiple instances of TopHat running on different samples or on behalf of different users, thus optimizing the overall execution time and enabling a flexible multi-user environmen

LaRA 2: parallel and vectorized program for sequence–structure alignment of RNA sequences

Background The function of non-coding RNA sequences is largely determined by their spatial conformation, namely the secondary structure of the molecule, formed by Watson–Crick interactions between nucleotides. Hence, modern RNA alignment algorithms routinely take structural information into account. In order to discover yet unknown RNA families and infer their possible functions, the structural alignment of RNAs is an essential task. This task demands a lot of computational resources, especially for aligning many long sequences, and it therefore requires efficient algorithms that utilize modern hardware when available. A subset of the secondary structures contains overlapping interactions (called pseudoknots), which add additional complexity to the problem and are often ignored in available software. Results We present the SeqAn-based software LaRA 2 that is significantly faster than comparable software for accurate pairwise and multiple alignments of structured RNA sequences. In contrast to other programs our approach can handle arbitrary pseudoknots. As an improved re-implementation of the LaRA tool for structural alignments, LaRA 2 uses multi-threading and vectorization for parallel execution and a new heuristic for computing a lower boundary of the solution. Our algorithmic improvements yield a program that is up to 130 times faster than the previous version. Conclusions With LaRA 2 we provide a tool to analyse large sets of RNA secondary structures in relatively short time, based on structural alignment. The produced alignments can be used to derive structural motifs for the search in genomic databases

isomiR-SEA: An RNA-Seq analysis tool for miRNAs/isomiRs expression level profiling and miRNA-mRNA interaction sites evaluation

>Background: Massive parallel sequencing of transcriptomes, revealed the presence of many miRNAs and miRNAs variants named isomiRs with a potential role in several cellular processes through their interaction with a target mRNA. Many methods and tools have been recently devised to detect and quantify miRNAs from sequencing data. However, all of them are implemented on top of general purpose alignment methods, thus providing poorly accurate results and no information concerning isomiRs and conserved miRNA-mRNA interaction sites. >Results: To overcome these limitations we present a novel algorithm named isomiR-SEA, that is able to provide users with very accurate miRNAs expression levels and both isomiRs and miRNA-mRNA interaction sites precise classifications. Tags are mapped on the known miRNAs sequences thanks to a specialized alignment algorithm developed on top of biological evidence concerning miRNAs structure. Specifically, isomiR-SEA checks for miRNA seed presence in the input tags and evaluates, during all the alignment phases, the positions of the encountered mismatches, thus allowing to distinguish among the different isomiRs and conserved miRNA-mRNA interaction sites. >Conclusions: isomiR-SEA performances have been assessed on two public RNA-Seq datasets proving that the implemented algorithm is able to account for more reliable and accurate miRNAs expression levels with respect to those provided by two compared state of the art tools. Moreover, differently from the few methods currently available to perform isomiRs detection, the proposed algorithm implements the evaluation of isomiRs and conserved miRNA-mRNA interaction sites already in the first alignment phases, thus avoiding any additional filtering stages potentially responsible for the loss of useful information

LaRA 2: parallel and vectorized program for sequence–structure alignment of RNA sequences

Background The function of non-coding RNA sequences is largely determined by their spatial conformation, namely the secondary structure of the molecule, formed by Watson–Crick interactions between nucleotides. Hence, modern RNA alignment algorithms routinely take structural information into account. In order to discover yet unknown RNA families and infer their possible functions, the structural alignment of RNAs is an essential task. This task demands a lot of computational resources, especially for aligning many long sequences, and it therefore requires efficient algorithms that utilize modern hardware when available. A subset of the secondary structures contains overlapping interactions (called pseudoknots), which add additional complexity to the problem and are often ignored in available software. Results We present the SeqAn-based software LaRA 2 that is significantly faster than comparable software for accurate pairwise and multiple alignments of structured RNA sequences. In contrast to other programs our approach can handle arbitrary pseudoknots. As an improved re-implementation of the LaRA tool for structural alignments, LaRA 2 uses multi-threading and vectorization for parallel execution and a new heuristic for computing a lower boundary of the solution. Our algorithmic improvements yield a program that is up to 130 times faster than the previous version. Conclusions With LaRA 2 we provide a tool to analyse large sets of RNA secondary structures in relatively short time, based on structural alignment. The produced alignments can be used to derive structural motifs for the search in genomic databases

Predicting gene and protein expression levels from DNA and protein sequences with Perceiver

Background and objective: The functions of an organism and its biological processes result from the expression of genes and proteins. Therefore quantifying and predicting mRNA and protein levels is a crucial aspect of scientific research. Concerning the prediction of mRNA levels, the available approaches use the sequence upstream and downstream of the Transcription Start Site (TSS) as input to neural networks. The State-of-the-art models (e.g., Xpresso and Basenjii) predict mRNA levels exploiting Convolutional (CNN) or Long Short Term Memory (LSTM) Networks. However, CNN prediction depends on convolutional kernel size, and LSTM suffers from capturing long-range dependencies in the sequence. Concerning the prediction of protein levels, as far as we know, there is no model for predicting protein levels by exploiting the gene or protein sequences. Methods: Here, we exploit a new model type (called Perceiver) for mRNA and protein level prediction, exploiting a Transformer-based architecture with an attention module to attend to long-range interactions in the sequences. In addition, the Perceiver model overcomes the quadratic complexity of the standard Transformer architectures. This work's contributions are 1. DNAPerceiver model to predict mRNA levels from the sequence upstream and downstream of the TSS; 2. ProteinPerceiver model to predict protein levels from the protein sequence; 3. Protein&DNAPerceiver model to predict protein levels from TSS and protein sequences. Results: The models are evaluated on cell lines, mice, glioblastoma, and lung cancer tissues. The results show the effectiveness of the Perceiver-type models in predicting mRNA and protein levels. Conclusions: This paper presents a Perceiver architecture for mRNA and protein level prediction. In the future, inserting regulatory and epigenetic information into the model could improve mRNA and protein level predictions. The source code is freely available at https://github.com/MatteoStefanini/DNAPerceiver

W2WNet: a two-module probabilistic Convolutional Neural Network with embedded data cleansing functionality

Convolutional Neural Networks (CNNs) are supposed to be fed with only high-quality annotated datasets. Nonetheless, in many real-world scenarios, such high quality is very hard to obtain, and datasets may be affected by any sort of image degradation and mislabelling issues. This negatively impacts the performance of standard CNNs, both during the training and the inference phase. To address this issue we propose Wise2WipedNet (W2WNet), a new two-module Convolutional Neural Network, where a Wise module exploits Bayesian inference to identify and discard spurious images during the training, and a Wiped module takes care of the final classification while broadcasting information on the prediction confidence at inference time. The goodness of our solution is demonstrated on a number of public benchmarks addressing different image classification tasks, as well as on a real-world case study on histological image analysis. Overall, our experiments demonstrate that W2WNet is able to identify image degradation and mislabelling issues both at training and at inference time, with a positive impact on the final classification accuracy