Development of efficient De Bruijn graph-based algorithms for genome assembly

Programa Oficial de Doutoramento en Computación. 5009V01[Abstract] During the last two decades, thanks to the development of new sequencing techniques, the study of the genome has become very popular in order to discover the genetic variation present in both humans and other organisms. The predominant mode of genome analysis is through the assembly of reads in one or multiple chains for as long as possible. The most traditional way of assembly is the one that involves reads from a single genome. In this field, in the last decade, third-generation readings have emerged with new challenges for which there are no efficient solutions. The first contribution that has been made in this thesis is Compact-Flye, a tool for the efficient assembly of third-generation reads on the Flye algorithm. This tool is based on the ingenious use of compact data structures to improve typical assembly steps such as counting and indexing k-mers. Apart from the assembly of a genome, there are techniques that seek to assemble all the genomes contained in a given sample. This assembly is known as multiple sequence assembly or haplotype reconstruction, a subject also treated in this thesis. Our first approach to solving this has been viaDBG, which is the first solution based on de Bruijn graphs that offers results comparable to current techniques in viral genome assembly while maintaining the efficiency of these graphs. Our second contribution is ViQUF, which is a natural improvement on its predecessor. ViQUF completely changes the algorithm of viaDBG but continues to be based on the same structures, although with some variations that allow it not only to improve results in terms of time and quality, but also to provide additionalinformation such as an estimate of the relative presence of each species in the sample.[Resumen] Durante las últimas dos décadas, gracias al desarrollo de nuevas técnias secuenciación, el estudio del genoma ha ganado mucha popularidad de cara a conocer la variación genética presente tanto seres humanos como otros organismos. El modo predominante de análisis del genoma es a través del ensamblaje de lecturas en una o múltiples cadenas lo más largas posibles. La manera más tradicional de ensamblaje es el que implica lecturas provenientes de un solo genoma. En este campo, en la última década han surgido las lecturas de tercera generación con nuevos retos para los que no existen soluciones eficientes. La primera aportación que se ha realizado en esta tesis es Compact-Flye una herramienta para el ensamblaje eficiente de lecturas de tercera generación sobre el algoritmo Flye. Esta herramienta está basada en el uso igenioso de estructuras compactas de datos para mejorar etapas típicas del ensamblaje como el conteo e indexación de k-mers. Al margen del ensamblaje de un genoma existen técnicas que buscan ensamblar todos los genomas contenidos en una muestra determinada. Este ensamblaje es conocido como ensamblaje múltiple de secuencias o reconstrucción de haplotipos, tema también tratado en esta tesis. Nuestra primera aproximación para la resolución de este ha sido viaDBG, que es la primera solución basada en grafos de de Bruijn que ofrece resultados comparables a las técnicas vigentes en ensamblaje de genomas víricos, mientras que mantiene la eficiencia de estos grafos. Nuestra segunda aportación es ViQUF, que es una mejora natural de su predecesor. ViQUF cambia totalmente la algoritmia de viaDBG, pero sigue cimentándose en las mismas estructuras aunque con alguna variación que le permite no solo mejorar resultados en tiempo y calidad. Sino que además le permite aportar más información como estimaciones relativa de cada especie en la muestra.[Resumo] Durante as dúas últimas décadas, grazas ao desenvolvemento de novas técnicas de secuenciación, o estudo do xenoma fíxose moi popular para descubrir a variación xenética presente tanto nos humanos como noutros organismos. O modo predominante de análise do xenoma é a través da ensamblaxe de lecturas nunha ou varias cadeas o maior tempo posible. A forma máis tradicional de ensamblar é a que implica lecturas dun só xenoma. Neste campo, na última década xurdiron lecturas de terceira xeración con novos retos para os que non existen solucións eficientes. A primeira contribución que se fixo nesta tese é Compact-Flye, unha ferramenta para a montaxe eficiente de lecturas de terceira xeración sobre o algoritmo Flye. Esta ferramenta baséase no uso intelixente de estruturas de datos compactas para mellorar os pasos típicos de montaxe, como contar e indexar k-mers. Ademais da montaxe dun xenoma, existen técnicas que buscan ensamblar todos os xenomas contidos nunha determinada mostra. Este conxunto coñécese como conxunto de secuencias múltiples ou reconstrución de haplotipos, tema tamén tratado nesta tesis. O noso primeiro enfoque para resolver isto foi viaDBG, que é a primeira solución baseada en gráficos de Bruijn que ofrece resultados comparables ás técnicas actuais de ensamblaxe de xenoma viral, mantendo a eficiencia destes gráficos. A nosa segunda incorporación é ViQUF, que é unha mellora natural con respecto ao seu predecesor. ViQUF cambia completamente o algoritmo de viaDBG pero segue baseándose nas mesmas estruturas, aínda que con algunha variación que lle permite non só mellorar os resultados en tempo e calidade. Pero tamén permite achegar máis información como estimacións relativas de cada especie da mostra.Xunta de Galicia; ED431G 2019/01Xunta de Galicia; ED431C 2021/53Xunta de Galicia; IG240.2020.1.185Xunta de Galicia; IN852A 2018/14Quiero agradecer al Centro de Investigación de Galicia “CITIC”, financiado por la Xunta de Galicia y la Unión Europea (European Regional Development Fund- Galicia 2014-2020 Program), con la beca ED431G 2019/01. También agradecer a la Xunta de Galicia/FEDER-UE que ha financiado esta tesis a través de las becas [ED431C 2021/53; IG240.2020.1.185; IN852A 2018/14]; al Ministerio de Ciencia e Innovación con las becas [TIN2016- 78011-C4-1-R; FPU17/02742; PID2019-105221RB-C41; PID2020-114635RB-I00]; y a la academia de Finlandia [grants 308030 and 323233 (LS)]

Inference of viral quasispecies with a paired de Bruijn graph

Motivation: RNA viruses exhibit a high mutation rate and thus they exist in infected cells as a population of closely related strains called viral quasispecies. The viral quasispecies assembly problem asks to characterize the quasispecies present in a sample from high-throughput sequencing data. We study the de novo version of the problem, where reference sequences of the quasispecies are not available. Current methods for assembling viral quasispecies are either based on overlap graphs or on de Bruijn graphs. Overlap graph-based methods tend to be accurate but slow, whereas de Bruijn graph-based methods are fast but less accurate. Results: We present viaDBG, which is a fast and accurate de Bruijn graph-based tool for de novo assembly of viral quasispecies. We first iteratively correct sequencing errors in the reads, which allows us to use large k-mers in the de Bruijn graph. To incorporate the paired-end information in the graph, we also adapt the paired de Bruijn graph for viral quasispecies assembly. These features enable the use of long-range information in contig construction without compromising the speed of de Bruijn graph-based approaches. Our experimental results show that viaDBG is both accurate and fast, whereas previous methods are either fast or accurate but not both. In particular, viaDBG has comparable or better accuracy than SAVAGE, while being at least nine times faster. Furthermore, the speed of viaDBG is comparable to PEHaplo but viaDBG is able to retrieve also low abundance quasispecies, which are often missed by PEHaplo.Peer reviewe

Inferring Genomic Sequences

Recent advances in next generation sequencing have provided unprecedented opportunities for high-throughput genomic research, inexpensively producing millions of genomic sequences in a single run. Analysis of massive volumes of data results in a more accurate picture of the genome complexity and requires adequate bioinformatics support. We explore computational challenges of applying next generation sequencing to particular applications, focusing on the problem of reconstructing viral quasispecies spectrum from pyrosequencing shotgun reads and problem of inferring informative single nucleotide polymorphisms (SNPs), statistically covering genetic variation of a genome region in genome-wide association studies. The genomic diversity of viral quasispecies is a subject of a great interest, particularly for chronic infections, since it can lead to resistance to existing therapies. High-throughput sequencing is a promising approach to characterizing viral diversity, but unfortunately standard assembly software cannot be used to simultaneously assemble and estimate the abundance of multiple closely related (but non-identical) quasispecies sequences. Here, we introduce a new Viral Spectrum Assembler (ViSpA) for inferring quasispecies spectrum and compare it with the state-of-the-art ShoRAH tool on both synthetic and real 454 pyrosequencing shotgun reads from HCV and HIV quasispecies. While ShoRAH has an advanced error correction algorithm, ViSpA is better at quasispecies assembling, producing more accurate reconstruction of a viral population. We also foresee ViSpA application to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations. Due to the large data volume in genome-wide association studies, it is desirable to find a small subset of SNPs (tags) that covers the genetic variation of the entire set. We explore the trade-off between the number of tags used per non-tagged SNP and possible overfitting and propose an efficient 2LR-Tagging heuristic

Methods for Viral Intra-Host and Inter-Host Data Analysis for Next-Generation Sequencing Technologies

The deep coverage offered by next-generation sequencing (NGS) technology has facilitated the reconstruction of intra-host RNA viral populations at an unprecedented level of detail. However, NGS data requires sophisticated analysis dealing with millions of error-prone short reads. This dissertation will first review the challenges and methods for viral NGS genomic data analysis in the NGS era. Second, it presents a software tool CliqueSNV for inferring viral quasispecies based on extracting pairs of statistically linked mutations from noisy reads, which effectively reduces sequencing noise and enables identifying minority haplotypes with a frequency below the sequencing error rate. Finally, the dissertation describes algorithms VOICE and MinDistB for inference of relatedness between viral samples, identification of transmission clusters, and sources of infection

Computational methods for understanding genetic variations from next generation sequencing data

Studies of human genetic variation reveal critical information about genetic and complex diseases such as cancer, diabetes and heart disease, ultimately leading towards improvements in health and quality of life. Moreover, understanding genetic variations in viral population is of utmost importance to virologists and helps in search for vaccines. Next-generation sequencing technology is capable of acquiring massive amounts of data that can provide insight into the structure of diverse sets of genomic sequences. However, reconstructing heterogeneous sequences is computationally challenging due to the large dimension of the problem and limitations of the sequencing technology.This dissertation is focused on algorithms and analysis for two problems in which we seek to characterize genetic variations: (1) haplotype reconstruction for a single individual, so-called single individual haplotyping (SIH) or haplotype assembly problem, and (2) reconstruction of viral population, the so-called quasispecies reconstruction (QSR) problem. For the SIH problem, we have developed a method that relies on a probabilistic model of the data and employs the sequential Monte Carlo (SMC) algorithm to jointly determine type of variation (i.e., perform genotype calling) and assemble haplotypes. For the QSR problem, we have developed two algorithms. The first algorithm combines agglomerative hierarchical clustering and Bayesian inference to reconstruct quasispecies characterized by low diversity. The second algorithm utilizes tensor factorization framework with successive data removal to reconstruct quasispecies characterized by highly uneven frequencies of its components. Both algorithms outperform existing methods in both benchmarking tests and real data.Electrical and Computer Engineerin

Efficient Minimum Flow Decomposition via Integer Linear Programming

Extended version of RECOMB 2022 paperMinimum flow decomposition (MFD) is an NP-hard problem asking to decompose a network flow into a minimum set of paths (together with associated weights). Variants of it are powerful models in multiassembly problems in Bioinformatics, such as RNA assembly. Owing to its hardness, practical multiassembly tools either use heuristics or solve simpler, polynomial time-solvable versions of the problem, which may yield solutions that are not minimal or do not perfectly decompose the flow. Here, we provide the first fast and exact solver for MFD on acyclic flow networks, based on Integer Linear Programming (ILP). Key to our approach is an encoding of all the exponentially many solution paths using only a quadratic number of variables. We also extend our ILP formulation to many practical variants, such as incorporating longer or paired-end reads, or minimizing flow errors. On both simulated and real-flow splicing graphs, our approach solves any instance inPeer reviewe