Quantitative measures for the management and comparison of annotated genomes

<p>Abstract</p> <p>Background</p> <p>The ever-increasing number of sequenced and annotated genomes has made management of their annotations a significant undertaking, especially for large eukaryotic genomes containing many thousands of genes. Typically, changes in gene and transcript numbers are used to summarize changes from release to release, but these measures say nothing about changes to individual annotations, nor do they provide any means to identify annotations in need of manual review.</p> <p>Results</p> <p>In response, we have developed a suite of quantitative measures to better characterize changes to a genome's annotations between releases, and to prioritize problematic annotations for manual review. We have applied these measures to the annotations of five eukaryotic genomes over multiple releases – <it>H. sapiens</it>, <it>M. musculus</it>, <it>D. melanogaster</it>, <it>A. gambiae</it>, and <it>C. elegans</it>.</p> <p>Conclusion</p> <p>Our results provide the first detailed, historical overview of how these genomes' annotations have changed over the years, and demonstrate the usefulness of these measures for genome annotation management.</p

Genome Assembly Has a Major Impact on Gene Content: A Comparison of Annotation in Two Bos Taurus Assemblies

Gene and SNP annotation are among the first and most important steps in analyzing a genome. As the number of sequenced genomes continues to grow, a key question is: how does the quality of the assembled sequence affect the annotations? We compared the gene and SNP annotations for two different Bos taurus genome assemblies built from the same data but with significant improvements in the later assembly. The same annotation software was used for annotating both sequences. While some annotation differences are expected even between high-quality assemblies such as these, we found that a staggering 40% of the genes (>9,500) varied significantly between assemblies, due in part to the availability of new gene evidence but primarily to genome mis-assembly events and local sequence variations. For instance, although the later assembly is generally superior, 660 protein coding genes in the earlier assembly are entirely missing from the later genome's annotation, and approximately 3,600 (15%) of the genes have complex structural differences between the two assemblies. In addition, 12–20% of the predicted proteins in both assemblies have relatively large sequence differences when compared to their RefSeq models, and 6–15% of bovine dbSNP records are unrecoverable in the two assemblies. Our findings highlight the consequences of genome assembly quality on gene and SNP annotation and argue for continued improvements in any draft genome sequence. We also found that tracking a gene between different assemblies of the same genome is surprisingly difficult, due to the numerous changes, both small and large, that occur in some genes. As a side benefit, our analyses helped us identify many specific loci for improvement in the Bos taurus genome assembly

First whole-genome sequence and assembly of the Ecuadorian brown-headed spider monkey ( Ateles fusciceps fusciceps ), a critically endangered species, using Oxford Nanopore Technologies

The Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps) is currently considered one of the most endangered primates in the world and is classified as critically endangered [International union for conservation of nature (IUCN)]. It faces multiple threats, the most significant one being habitat loss due to deforestation in western Ecuador. Genomic tools are keys for the management of endangered species, but this requires a reference genome, which until now was unavailable for A. f. fusciceps. The present study reports the first whole-genome sequence and assembly of A. f. fusciceps generated using Oxford Nanopore long reads. DNA was extracted from a subadult male, and libraries were prepared for sequencing following the Ligation Sequencing Kit SQK-LSK112 workflow. Sequencing was performed using a MinION Mk1C sequencer. The sequencing reads were processed to generate a genome assembly. Two different assemblers were used to obtain draft genomes using raw reads, of which the Flye assembly was found to be superior. The final assembly has a total length of 2.63 Gb and contains 3,861 contigs, with an N50 of 7,560,531 bp. The assembly was analyzed for annotation completeness based on primate ortholog prediction using a high-resolution database, and was found to be 84.3% complete, with a low number of duplicated genes indicating a precise assembly. The annotation of the assembly predicted 31,417 protein-coding genes, comparable with other mammal assemblies. A reference genome for this critically endangered species will allow researchers to gain insight into the genetics of its populations and thus aid conservation and management efforts of this vulnerable species

Ranked Choice Voting for Representative Transcripts with TRaCE

Genome sequencing projects annotate protein-coding gene models with multiple transcripts, aiming to represent all of the available transcript evidence. However, downstream analyses often operate on only one representative transcript per gene locus, sometimes known as the canonical transcript. To choose canonical transcripts, TRaCE (Transcript Ranking and Canonical Election) holds an ‘election’ in which a set of RNA-seq samples rank transcripts by annotation edit distance. These sample-specific votes are tallied along with other criteria such as protein length and InterPro domain coverage. The winner is selected as the canonical transcript, but the election proceeds through multiple rounds of voting to order all the transcripts by relevance. Based on the set of expression data provided, TRaCE can identify the most common isoforms from a broad expression atlas or prioritize alternative transcripts expressed in specific contexts

Doctor of Philosophy

dissertationWhole genome sequencing projects have expanded our understanding of evolution, organism development, and human disease. Now advances in secondgeneration technologies are making whole genome sequencing routine even for small laboratories. However, advances in annotation technology have not kept pace with genome sequencing, and annotation has become the major bottleneck for many genome projects (especially those with limited bioinformatics expertise). At the same time, challenges associated with genomics research extend beyond merely annotating genomes, as annotations must be subjected to diverse downstream analyses, the complexities of which can confound smaller research groups. Additionally, with improvements in genome assembly and the wide availability of next generation transcriptome data (mRNA-seq), researchers have the opportunity to re-annotate previously published genomes, which creates new difficulties for data integration and management that are not well addressed by existing tools. In response to the challenges facing second-generation genome projects, I have developed the annotation pipeline MAKER2 together with accessory software for downstream analysis and data management. The MAKER2 annotation pipeline finds repeats within a genome, aligns ESTs and cDNAs, identifies sites of protein homology, and produces database-ready gene annotations in association with supporting evidence. However MAKER2 can go beyond structural annotation to identify and integrate functional annotations. MAKER2 also provides researchers iv with the capability to re-annotate legacy genome datasets and to incorporate mRNAseq. Additionally, MAKER2 supports distributed parallelization on computer clusters, thus providing a scalable solution for datasets of any size. Annotations produced by MAKER2 can be directly loaded into many popular downstream annotation analysis and management tools from the Generic Model Organism Database Project. By using MAKER2 with these tools, research groups can quickly build genome annotations, perform analyses, and distribute their data to the wider scientific community. Here I describe the internal architecture of MAKER2, and document its computational capabilities. I also describe my work to annotate and analyze eight emerging model organism genomes in collaboration with their associated genome projects. Thus, in the course of my thesis work, I have addressed a specific need within the scientific community for easy-to-use annotation and analysis tools while also expanding our understanding of evolution and biology

Whole genome sequencing of the Asian Arowana (Scleropages formosus) provides insights into the evolution of ray-finned fishes

The Asian arowana (Scleropages formosus) is of commercial importance, conservation concern, and is a representative of one of the oldest lineages of ray-finned fish, the Osteoglossomorpha. To add to genomic knowledge of this species and the evolution of teleosts, the genome of a Malaysian specimen of arowana was sequenced. A draft genome is presented consisting of 42,110 scaffolds with a total size of 708 Mb (2.85% gaps) representing 93.95% of core eukaryotic genes. Using a k-mer-based method, a genome size of 900 Mb was also estimated. We present an update on the phylogenomics of fishes based on a total of 27 species (23 fish species and 4 tetrapods) using 177 orthologous proteins (71,360 amino acid sites), which supports established relationships except that arowana is placed as the sister lineage to all teleost clades (Bayesian posterior probability 1.00, bootstrap replicate 93%), that evolved after the teleost genome duplication event rather than the eels (Elopomorpha). Evolutionary rates are highly heterogeneous across the tree with fishes represented by both slowly and rapidly evolving lineages. A total of 94 putative pigment genes were identified, providing the impetus for development of molecular markers associated with the spectacular colored phenotypes found within this species

The genome and developmental transcriptome of the strongylid nematode Haemonchus contortus

Background: The barber's pole worm, Haemonchus contortus, is one of the most economically important parasites of small ruminants worldwide. Although this parasite can be controlled using anthelmintic drugs, resistance against most drugs in common use has become a widespread problem. We provide a draft of the genome and the transcriptomes of all key developmental stages of H. contortus to support biological and biotechnological research areas of this and related parasites. Results: The draft genome of H. contortus is 320 Mb in size and encodes 23,610 protein-coding genes. On a fundamental level, we elucidate transcriptional alterations taking place throughout the life cycle, characterize the parasite's gene silencing machinery, and explore molecules involved in development, reproduction, host-parasite interactions, immunity, and disease. The secretome of H. contortus is particularly rich in peptidases linked to blood-feeding activity and interactions with host tissues, and a diverse array of molecules is involved in complex immune responses. On an applied level, we predict drug targets and identify vaccine molecules. Conclusions: The draft genome and developmental transcriptome of H. contortus provide a major resource to the scientific community for a wide range of genomic, genetic, proteomic, metabolomic, evolutionary, biological, ecological, and epidemiological investigations, and a solid foundation for biotechnological outcomes, including new anthelmintics, vaccines and diagnostic tests. This first draft genome of any strongylid nematode paves the way for a rapid acceleration in our understanding of a wide range of socioeconomically important parasites of one of the largest nematode orders

Doctor of Philosophy

dissertationThe MAKER genome annotation and curation software tool was developed in response to increased demand for genome annotation services, secondary to decreased genome sequencing costs. MAKER currently has over 1000 registered users throughout the world. This wide adoption of MAKER has uncovered the need for additional functionalities. Here I addressed moving MAKER into the domain of plant annotation, expanding MAKER to include new methods of gene and noncoding RNA annotation, and improving usability of MAKER through documentation and community outreach. To move MAKER into the plant annotation domain, I benchmarked MAKER on the well-annotated Arabidopsis thaliana genome. MAKER performs well on the Arabidopsis genome in de novo genome annotation and was able to improve the current TAIR10 gene models by incorporating mRNA-seq data not available during the original annotation efforts. In addition to this benchmarking, I annotated the genome of the sacred lotus Nelumbo Nucifera. I enabled noncoding RNA annotation in MAKER by adding the ability for MAKER to run and process the outputs of tRNAscan-SE and snoscan. These functionalities were tested on the Arabidopsis genome and used MAKER to annotate tRNAs and snoRNAs in Zea mays. The resulting version of MAKER was named MAKER-P. I added the functionality of a combiner by adding EVidence Modeler to the MAKER code base. iv As the number of MAKER users has grown, so have the help requests sent to the MAKER developers list. Motivated by the belief that improving the MAKER documentation would obviate the need for many of these requests, I created a media wiki that was linked to the MAKER download page, and the MAKER developers list was made searchable. Additionally I have written a unit on genome annotation using MAKER for Current Protocols in Bioinformatics. In response to these efforts I have seen a corresponding decrease in help requests, even though the number of registered MAKER users continues to increase. Taken together these products and activities have moved MAKER into the domain of plant annotation, expanded MAKER to include new methods of gene and noncoding RNA annotation, and improved the usability of MAKER through documentation and community outreach

Uso de datos de transcriptómica para la anotación de genes en el genoma de pacú (Piaractus mesopotamicus)

En Argentina, la producción de pacú (Piaractus mesopotamicus) ocupa el primer lugar dentro de las producciones por piscicultura de especies nativas. A pesar de la importancia de la misma, existe poca información genómica sobre esta especie, lo cual limita el desarrollo de tecnología y de programas de mejoramiento genético. En el Laboratorio Mixto de Biotecnología Acuática fueron ensamblados recientemente un genoma de referencia para hembra y uno para macho a partir de la tecnología de secuenciación corta de Illumina. Sin embargo, los mismos aún no han sido anotados. Profundizar el análisis de dichos genomas permitirá no solo aumentar el conocimiento sobre diferentes aspectos de la biología del pacú, sino que también dará lugar al diseño y desarrollo de nuevos proyectos. Es por ello que el objetivo de este trabajo fue contribuir a la anotación de dichos genomas utilizando datos de transcriptómica disponibles para esta especie. En el presente trabajo se utilizaron secuencias de un transcriptoma de músculo de pacú provenientes de un repositorio público. Se realizó un análisis de calidad de las secuencias crudas y luego se ensamblaron de manera conjunta usando el programa Trinity. El análisis del ensamblado indicó que el mismo es de alta calidad, con un alto aprovechamiento de las secuencias crudas (98.84 %) y alta presencia (81.4%) de ortólogos de ciertos genes que son universales, se expresan persistentemente y se presentan casi exclusivamente como copias únicas en el genoma. Para la anotación funcional se combinaron datos de predicción de secuencias proteicas, análisis de homologías de secuencias y predicción de dominios, de péptidos señales y de familias. Al anotar el transcriptoma se encontraron 64.111 genes e isoformas, indicando una perspectiva de alta utilidad en el uso de estas secuencias para anotar los genomas. Los genomas obtenidos para un macho y una hembra de pacú a partir de tecnología Illumina se encuentran altamente fragmentados. Esto trajo como consecuencia una gran dificultad computacional en el proceso de anotación de los mismos, a pesar de trabajar en un servidor con un alto poder de cómputo. Por esto, solo se llevó a cabo una prueba de anotación de un fragmento de cada genoma. El genoma de la hembra se procesó en un 38%, anotando 5.970 genes, con un largo promedio de 4.660,44 bases. El genoma del macho, en cambio, se procesó en un 23%, anotando 4.182 genes, con un largo promedio de 3.953,42 bases A pesar de ser resultados preliminares los mismos son alentadores, ya que presentan altos niveles de calidad según el parámetro AED.In Argentina, the production of pacú (Piaractus mesopotamicus) occupies the first place within the productions by fish farming of native species. Despite its importance, there is little genomic information on this species, which limits the development of technology and breeding programs. A female and a male reference genome were recently assembled at the Joint Aquatic Biotechnology Laboratory using Illumina short sequencing technology. However, they have not yet been annotated. Deepening the analysis of these genomes will allow not only to increase knowledge about different aspects of the biology of the pacú, but will also lead to the design and development of new projects. That is why the aim of this work was to contribute to In the present work, sequences of a pacú muscle transcriptome from a public repository were used. Quality analysis of the raw sequences was performed and these were then assembled together using the Trinity program. The analysis of the assembly indicated that it is of high quality, with a high use of raw sequences (98.84%) and a high presence (81.4%) of orthologs of certain genes that are universal, are persistently expressed and are presented almost exclusively as unique copies in the genome. Data from protein sequence prediction, sequence homology analysis, and domain, signal peptide, and family prediction were combined for functional annotation. Annotating the transcriptome found 64,111 genes and isoforms, indicating a prospect of high utility in using these sequences to annotate genomes. Genomes obtained for one male and one female pacú from Illumina technology are highly fragmented. This resulted in great computational difficulty in the annotation process, despite working on a server with high computing power. For this reason, only one annotation test of a fragment of each genome was carried out. The female genome was 38% processed, scoring 5,970 genes, with an average length of 4,660.44 bases. The male genome, on the other hand, was processed in 23%, annotating 4,182 genes, with an average length of 3,953.42 bases. Despite being preliminary results, they are encouraging, since they present high levels of quality according to the AED parameter.Fil: Apellido, Nombre. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaFil: Posner, Victoria María. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; Argentin

Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source. The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log2 fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose