Next generation sequencing of SARS-CoV-2 genomes: challenges, applications and opportunities.

Various next generation sequencing (NGS) based strategies have been successfully used in the recent past for tracing origins and understanding the evolution of infectious agents, investigating the spread and transmission chains of outbreaks, as well as facilitating the development of effective and rapid molecular diagnostic tests and contributing to the hunt for treatments and vaccines. The ongoing COVID-19 pandemic poses one of the greatest global threats in modern history and has already caused severe social and economic costs. The development of efficient and rapid sequencing methods to reconstruct the genomic sequence of SARS-CoV-2, the etiological agent of COVID-19, has been fundamental for the design of diagnostic molecular tests and to devise effective measures and strategies to mitigate the diffusion of the pandemic. Diverse approaches and sequencing methods can, as testified by the number of available sequences, be applied to SARS-CoV-2 genomes. However, each technology and sequencing approach has its own advantages and limitations. In the current review, we will provide a brief, but hopefully comprehensive, account of currently available platforms and methodological approaches for the sequencing of SARS-CoV-2 genomes. We also present an outline of current repositories and databases that provide access to SARS-CoV-2 genomic data and associated metadata. Finally, we offer general advice and guidelines for the appropriate sharing and deposition of SARS-CoV-2 data and metadata, and suggest that more efficient and standardized integration of current and future SARS-CoV-2-related data would greatly facilitate the struggle against this new pathogen. We hope that our 'vademecum' for the production and handling of SARS-CoV-2-related sequencing data, will contribute to this objective.

Comparative Genomics Reveals Early Emergence and Biased Spatiotemporal Distribution of SARS-CoV-2.

Effective systems for the analysis of molecular data are fundamental for monitoring the spread of infectious diseases and studying pathogen evolution. The rapid identification of emerging viral strains, and/or genetic variants potentially associated with novel phenotypic features is one of the most important objectives of genomic surveillance of human pathogens and represents one of the first lines of defense for the control of their spread. During the COVID 19 pandemic, several taxonomic frameworks have been proposed for the classification of SARS-Cov-2 isolates. These systems, which are typically based on phylogenetic approaches, represent essential tools for epidemiological studies as well as contributing to the study of the origin of the outbreak. Here, we propose an alternative, reproducible, and transparent phenetic method to study changes in SARS-CoV-2 genomic diversity over time. We suggest that our approach can complement other systems and facilitate the identification of biologically relevant variants in the viral genome. To demonstrate the validity of our approach, we present comparative genomic analyses of more than 175,000 genomes. Our method delineates 22 distinct SARS-CoV-2 haplogroups, which, based on the distribution of high-frequency genetic variants, fall into four major macrohaplogroups. We highlight biased spatiotemporal distributions of SARS-CoV-2 genetic profiles and show that seven of the 22 haplogroups (and of all of the four haplogroup clusters) showed a broad geographic distribution within China by the time the outbreak was widely recognized-suggesting early emergence and widespread cryptic circulation of the virus well before its isolation in January 2020. General patterns of genomic variability are remarkably similar within all major SARS-CoV-2 haplogroups, with UTRs consistently exhibiting the greatest variability, with s2m, a conserved secondary structure element of unknown function in the 3'-UTR of the viral genome showing evidence of a functional shift. Although several polymorphic sites that are specific to one or more haplogroups were predicted to be under positive or negative selection, overall our analyses suggest that the emergence of novel types is unlikely to be driven by convergent evolution and independent fixation of advantageous substitutions, or by selection of recombined strains. In the absence of extensive clinical metadata for most available genome sequences, and in the context of extensive geographic and temporal biases in the sampling, many questions regarding the evolution and clinical characteristics of SARS-CoV-2 isolates remain open. However, our data indicate that the approach outlined here can be usefully employed in the identification of candidate SARS-CoV-2 genetic variants of clinical and epidemiological importance.

LC-HRMS-Database Screening Metrics for Rapid Prioritization of Samples to Accelerate the Discovery of Structurally New Natural Products.

In order to accelerate the isolation and characterization of structurally new or novel secondary metabolites, it is crucial to develop efficient strategies that prioritize samples with greatest promise early in the workflow so that resources can be utilized in a more efficient and cost-effective manner. We have developed a metrics-based prioritization approach using exact LC-HRMS, which uses data for 24 618 marine natural products held in the PharmaSea database. Each sample was evaluated and allocated a metric score by a software algorithm based on the ratio of new masses over the total (sample novelty), ratio of known masses over the total (chemical novelty), number of peaks above a defined peak area threshold (sample complexity), and peak area (sample diversity). Samples were then ranked and prioritized based on these metric scores. To validate the approach, eight marine sponges and six tunicate samples collected from the Fiji Islands were analyzed, metric scores calculated, and samples targeted for isolation and characterization of new compounds. Structures of new compounds were elucidated by spectroscopic techniques, including 1D and 2D NMR, MS, and MS/MS. Structures were confirmed by computer-assisted structure elucidation methods (CASE) using the ACD/Structure Elucidator Suite.

Extensive mitochondrial gene rearrangements in Ctenophora: insights from benthic Platyctenida.

BACKGROUND: Complete mitochondrial (mt) genomes have been sequenced for thousands of animals and represent a molecule of choice for many evolutionary studies. Nevertheless, some animal groups have remained under-sampled. Ctenophora (comb jellies) is one such example, with only two complete mt sequences determined hitherto for this phylum, which encompasses ca. 150-200 described species. This lack of data derives from the extremely fast mt evolutionary rate in this lineage, complicating primer design and DNA amplification. Indeed, in the two ctenophore mt genomes sequenced to date, i.e. those of Mnemiopsis leidyi (order Lobata) and Pleurobrachia bachei (order Cydippida), both rRNA and protein coding genes exhibit an extraordinary size reduction and have highly derived sequences. Additionally, all tRNAs, and the atp6 and atp8 genes are absent. In order to determine whether these characteristics are shared by other ctenophores, we obtained the complete mt genomes of three benthic ctenophores belonging to the so far unsampled order of Platyctenida: Coeloplana loyai, Coeloplana yulianicorum and Vallicula multiformis. RESULTS: The mt genomes of benthic ctenophores reveal the same peculiarities found in Mnemiopsis and Pleurobrachia, demonstrating that the fast evolutionary rate is a general trait of the ctenophore mt genomes. Our results also indicate that this high evolutionary rate not only affects the nucleotide substitution but also gene rearrangements. Indeed, gene order was highly rearranged among representatives of the different taxonomic orders in which it was close to random, but also quite variable within Platyctenida, in which the genera Coeloplana and Vallicula share only four conserved synteny blocks. However, the two congeneric Coeloplana species display exactly the same gene order. Because of the extreme evolutionary rate, our phylogenetic analyses were unable to resolve the phylogenetic position of ctenophores within metazoans or the relationships among the different Ctenophora orders. Comparative sequence-analyses allowed us to correct the annotation of the Pleurobrachia mt genome, confirming the absence of tRNAs, the presence of both rRNA genes, and the existence of a reassignment of codon TGA from tryptophan to serine for this species. CONCLUSIONS: Since Platyctenida is an early diverging lineage among Ctenophora, our findings suggest that the mt traits described above are ancestral characteristics of this phylum.

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians.

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

Back to solitude: Solving the phylogenetic position of the Diazonidae using molecular and developmental characters.

The order Aplousobranchia (Chordata, Ascidiacea) contains approximately 1500 species distributed worldwide. Their phylogeny, however, remains unclear, with unresolved family relationships. While most Aplousobranchia are colonial, debates exist concerning the phylogenetic position of families such as the Diazonidae and Cionidae, which exhibit a solitary lifestyle and share morphological characteristics with both Aplousobranchia and Phlebobranchia orders. To clarify the phylogenetic position of the Diazonidae and Cionidae, we determined the complete mitochondrial sequence of the solitary diazonid Rhopalaea idoneta. The phylogenetic reconstruction based on the 13 mitochondrial protein coding genes strongly supports a positioning of Diazonidae well-nested within the Aplousobranchia rather than a positioning as a sister clade of the Aplousobranchia. In addition, we examined the regenerative ability of R. idoneta. Similar to colonial Aplousobranchia, R. idoneta was found to be able to completely regenerate its thorax. Ciona, also known to possess high regenerative abilities, is the Aplousobranchia sister clade rather than a member of the Phlebobranchia. Our results thus indicate that the colonial lifestyle was acquired in the Aplousobranchia, starting from a Ciona-like solitary ancestor and secondarily lost in Diazonidae representatives such as Rhopalaea. The solitary lifestyle of Rhopalaea is thus a derived characteristic rather than an ancestral trait.

MitoZoa 2.0: a database resource and search tools for comparative and evolutionary analyses of mitochondrial genomes in Metazoa.

The MITOchondrial genome database of metaZOAns (MitoZoa) is a public resource for comparative analyses of metazoan mitochondrial genomes (mtDNA) at both the sequence and genomic organizational levels. The main characteristics of the MitoZoa database are the careful revision of mtDNA entry annotations and the possibility of retrieving gene order and non-coding region (NCR) data in appropriate formats. The MitoZoa retrieval system enables basic and complex queries at various taxonomic levels using different search menus. MitoZoa 2.0 has been enhanced in several aspects, including: a re-annotation pipeline to check the correctness of protein-coding gene predictions; a standardized annotation of introns and of precursor ORFs whose functionality is post-transcriptionally recovered by RNA editing or programmed translational frameshifting; updates of taxon-related fields and a BLAST sequence similarity search tool. Database novelties and the definition of standard mtDNA annotation rules, together with the user-friendly retrieval system and the BLAST service, make MitoZoa a valuable resource for comparative and evolutionary analyses as well as a reference database to assist in the annotation of novel mtDNA sequences. MitoZoa is freely accessible at http://www.caspur.it/mitozoa.

Detection of ribonucleotides embedded in DNA by Nanopore sequencing.

Ribonucleotides represent the most common non-canonical nucleotides found in eukaryotic genomes. The sources of chromosome-embedded ribonucleotides and the mechanisms by which unrepaired rNMPs trigger genome instability and human pathologies are not fully understood. The available sequencing technologies only allow to indirectly deduce the genomic location of rNMPs. Oxford Nanopore Technologies (ONT) may overcome such limitation, revealing the sites of rNMPs incorporation in genomic DNA directly from raw sequencing signals. We synthesized two types of DNA molecules containing rNMPs at known or random positions and we developed data analysis pipelines for DNA-embedded ribonucleotides detection by ONT. We report that ONT can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-specific alterations in nucleotide alignment features, current intensity, and dwell time. We propose that ONT may be successfully employed to directly map rNMPs in genomic DNA and we suggest a strategy to build an ad hoc basecaller to analyse native genomes.

Bioinformatics in Italy: BITS 2012, the ninth annual meeting of the Italian Society of Bioinformatics.

The BITS2012 meeting, held in Catania on May 2-4, 2012, brought together almost 100 Italian researchers working in the field of Bioinformatics, as well as students in the same or related disciplines. About 90 original research works were presented either as oral communication or as posters, representing a landscape of Italian current research in bioinformatics. This preface provides a brief overview of the meeting and introduces the manuscripts that were accepted for publication in this supplement, after a strict and careful peer-review by an International board of referees.

Expression and phylogenetic analyses of the Gel/Gas proteins of Tuber melanosporum provide insights into the function and evolution of glucan remodeling enzymes in fungi.

The ß(1,3)-glucanosyltransferases of the GH72 family are redundant enzymes that are essential for the formation and dynamic remodeling of the fungal wall during different stages of the life cycle. Four putative genes encoding glycosylphosphatidylinositol (GPI)-anchored ß(1,3)-glucanosyltransferases, designated TmelGEL1, TmelGEL2, TmelGEL4 and TmelGAS4, have been annotated in the genome of Tuber melanosporum, an ectomycorrhizal fungus that also produces a hypogeous fruiting body (FB) of great commercial value (black truffle). This work focuses on the characterization and expression of this multigene family by taking advantage of a laser microdissection (LMD) technology that has been used to separate two distinct compartments in the FB, the hyphae and the asci containing the ascospores. Of the four genes, TmelGEL1 was the most up-regulated in the FB compared to the free-living mycelium. Inside the FB, the expression of TmelGEL1 was restricted to the hyphal compartment. A phylogenetic analysis of the Gel/Gas protein family of T. melanosporum was also carried out. A total of 237 GH72 proteins from 51 Ascomycotina and 3 Basidiomycota (outgroup) species were analyzed. The resulting tree provides insight into the evolution of the T. melanosporum proteins and identifies new GH72 paralogs/subfamilies. Moreover, it represents a starting point to formulate new hypotheses on the significance of the striking GH72 gene redundancy in fungal biology.

HaploCoV: unsupervised classification and rapid detection of novel emerging variants of SARS-CoV-2.

Accurate and timely monitoring of the evolution of SARS-CoV-2 is crucial for identifying and tracking potentially more transmissible/virulent viral variants, and implement mitigation strategies to limit their spread. Here we introduce HaploCoV, a novel software framework that enables the exploration of SARS-CoV-2 genomic diversity through space and time, to identify novel emerging viral variants and prioritize variants of potential epidemiological interest in a rapid and unsupervised manner. HaploCoV can integrate with any classification/nomenclature and incorporates an effective scoring system for the prioritization of SARS-CoV-2 variants. By performing retrospective analyses of more than 11.5 M genome sequences we show that HaploCoV demonstrates high levels of accuracy and reproducibility and identifies the large majority of epidemiologically relevant viral variants - as flagged by international health authorities - automatically and with rapid turn-around times.Our results highlight the importance of the application of strategies based on the systematic analysis and integration of regional data for rapid identification of novel, emerging variants of SARS-CoV-2. We believe that the approach outlined in this study will contribute to relevant advances to current and future genomic surveillance methods.

Amplicon-Based Microbiome Profiling: From Second- to Third-Generation Sequencing for Higher Taxonomic Resolution.

The 16S rRNA amplicon-based sequencing approach represents the most common and cost-effective strategy with great potential for microbiome profiling. The use of second-generation sequencing (NGS) technologies has led to protocols based on the amplification of one or a few hypervariable regions, impacting the outcome of the analysis. Nowadays, comparative studies are necessary to assess different amplicon-based approaches, including the full-locus sequencing currently feasible thanks to third-generation sequencing (TGS) technologies. This study compared three different methods to achieve the deepest microbiome taxonomic characterization: (a) the single-region approach, (b) the multiplex approach, covering several regions of the target gene/region, both based on NGS short reads, and (c) the full-length approach, which analyzes the whole length of the target gene thanks to TGS long reads. Analyses carried out on benchmark microbiome samples, with a known taxonomic composition, highlighted a different classification performance, strongly associated with the type of hypervariable regions and the coverage of the target gene. Indeed, the full-length approach showed the greatest discriminating power, up to species level, also on complex real samples. This study supports the transition from NGS to TGS for the study of the microbiome, even if experimental and bioinformatic improvements are still necessary.

Yamanaka Factors in the Budding Tunicate Botryllus schlosseri Show a Shared Spatio-Temporal Expression Pattern in Chordates.

In vertebrates, the four transcription factors Sox2, c-Myc, Pou5f1 and Klf4 are involved in the differentiation of several tissues during vertebrate embryogenesis; moreover, they are normally co-expressed in embryonic stem cells and play roles in pluripotency, self-renewal, and maintenance of the undifferentiated state in adult cells. The in vitro forced co-expression of these factors, named Yamanaka factors (YFs), induces pluripotency in human or mouse fibroblasts. Botryllus schlosseri is a colonial tunicate undergoing continuous stem cell-mediated asexual development, providing a valuable model system for the study of pluripotency in the closest living relatives of vertebrates. In this study, we identified B. schlosseri orthologs of human Sox2 and c-Myc genes, as well as the closest homologs of the vertebrate-specific Pou5f1 gene, through an in-depth evolutionary analysis of the YF gene families in tunicates and other deuterostomes. Then, we studied the expression of these genes during the asexual cycle of B. schlosseri using in situ hybridization in order to investigate their possible involvement in tissue differentiation and in pluripotency maintenance. Our results show a shared spatio-temporal expression pattern consistent with the reported functions of these genes in invertebrate and vertebrate embryogenesis. Moreover, Myc, SoxB1 and Pou3 were expressed in candidate stem cells residing in their niches, while Pou2 was found expressed exclusively in the immature previtellogenic oocytes, both in gonads and circulating in the colonial vascular system. Our data suggest that Myc, SoxB1 and Pou3 may be individually involved in the differentiation of the same territories seen in other chordates, and that, together, they may play a role in stemness even in this colonial ascidian.

Hypervariability of ascidian mitochondrial gene order: exposing the myth of deuterostome organelle genome stability.

The few sequenced mitochondrial (mt) genomes of the class Ascidiacea (Chordata, Tunicata), mostly belonging to congeneric species of the Phlebobranchia order, show extraordinary gene order rearrangements. In order to assess if this hypervariability in gene order is a general feature of Ascidiacea, we report here the gene arrangement of five ascidians belonging to the Aplousobranchia and Stolidobranchia orders. Our data show that Ascidiacea are characterized by: 1) extensive gene order rearrangements both within and between the three major lineages; 2) lack of significant similarities to the gene order of other deuterostomes; and 3) an extent of rearrangements comparable with that of Mollusca (especially the Gastropoda, Bivalvia, and Scaphopoda classes), a phylum with highly rearranged mtDNAs. The only conserved feature is the location of all genes on the same strand, which suggests that selective constraints are related to the mt transcription. Finally, a higher mobility of the tRNA genes is undetectable because of saturation effect, and only the partially conserved cox2-cob gene block seems to retain some phylogenetic signals.

Finding Aquaporins in Annelids: An Evolutionary Analysis and a Case Study.

Aquaporins (AQPs) are a family of membrane channels facilitating diffusion of water and small solutes into and out of cells. Despite their biological relevance in osmoregulation and ubiquitous distribution throughout metazoans, the presence of AQPs in annelids has been poorly investigated. Here, we searched and annotated Aqp sequences in public genomes and transcriptomes of annelids, inferred their evolutionary relationships through phylogenetic analyses and discussed their putative physiological relevance. We identified a total of 401 Aqp sequences in 27 annelid species, including 367 sequences previously unrecognized as Aqps. Similar to vertebrates, phylogenetic tree reconstructions clustered these annelid Aqps in four clades: AQP1-like, AQP3-like, AQP8-like and AQP11-like. We found no clear indication of the existence of paralogs exclusive to annelids; however, several gene duplications seem to have occurred in the ancestors of some Sedentaria annelid families, mainly in the AQP1-like clade. Three of the six Aqps annotated in Alitta succinea, an estuarine annelid showing high salinity tolerance, were validated by RT-PCR sequencing, and their similarity to human AQPs was investigated at the level of "key" conserved residues and predicted three-dimensional structure. Our results suggest a diversification of the structures and functions of AQPs in Annelida comparable to that observed in other taxa.

An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode.

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

Evaluating the Efficiency of DNA Metabarcoding to Analyze the Diet of Hippocampus guttulatus (Teleostea: Syngnathidae).

Seahorses are considered a flagship species for conservation efforts and due to their conservation status, improving knowledge on their dietary composition while applying a non-invasive approach, could be useful. Using Hippocampus guttulatus as a case study, the present study represents pioneering research into investigating the diet of seahorses by NGS-based DNA metabarcoding of fecal samples. The study developed and tested the protocol for fecal DNA metabarcoding during the feeding trials where captive seahorses were fed on a diet of known composition; the process was subsequently applied on fecal samples collected from wild individuals. The analysis of samples collected during the feeding trials indicated the reliability of the applied molecular approach by allowing the characterization of the effectively ingested prey. In the field study, among detected prey species, results revealed that the majority of the seahorse samples contained taxa such as Amphipoda, Decapoda, Isopoda, and Calanoida, while less common prey taxa were Gastropoda and Polyplacophora. As only a small amount of starting fecal material is needed and the sampling procedure is neither invasive nor lethal. The present study indicates DNA metabarcoding as useful for investigating seahorse diet and could help define management and conservation actions.

Assessment of orthologous splicing isoforms in human and mouse orthologous genes.

BACKGROUND: Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. RESULTS: As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. CONCLUSIONS: We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still maintaining the conventional definition of gene orthology, a new concept of "splicing orthology" can be defined at transcript level.

Mitogenomics reveals two cryptic species in Ciona intestinalis.

Individual mitochondrial genes or genomic features are commonly used as phylogenetic markers at many taxonomic levels. We used a mitogenomics approach to demonstrate the existence of two cryptic species in the ascidian Ciona intestinalis, a model chordate whose status as a single species has recently been questioned. Comprehensive comparative analysis of the mitochondrial genome of the two cryptic species revealed significant differences in gene order, size and number of noncoding regions, compositional features and divergence of protein-coding genes.

Phylogenetic comparison of huntingtin homologues reveals the appearance of a primitive polyQ in sea urchin.

Huntingtin is a completely soluble 3,144 amino acid (aa) protein characterized by the presence of an amino-terminal polymorphic polyglutamine (polyQ) tract, whose aberrant expansion causes the progressively neurodegenerative Huntington's disease (HD). Biological evidence indicates that huntingtin (htt) is beneficial to cells (particularly to brain neurons) and that loss of its neuronal function may contribute to HD. The exact protein domains involved in its neuroprotective function are unknown. Evolutionary analyses of htt primary aa have so far been limited to a few species, but its thorough assessment may help to clarify the functions emerging during evolution. We made an extensive comparative analysis of the available htt protein homologues from different organisms along the metazoan phylogenetic tree and defined the presence of 3 different conservative blocks corresponding to human htt aa 1-386 (htt1), 683-1,586 (htt2), and 2,437-3,078 (htt3), in which HEAT (Huntingtin, Elongator factor3, the regulatory A subunit of protein phosphatase 2A, and TOR1) repeats are well conserved. We also describe the cloning and sequencing of sea urchin htt mRNA, the oldest deuterostome homologue so far available. Multiple alignment shows the first appearance of a primitive polyQ in sea urchin, which predates an ancestral polyQ sequence in a nonchordate environment and defines the polyQ characteristic as being typical of the deuterostome branch. The fact that glutamines have conserved positions in deuterostomes and the polyQ size increases during evolution suggests that the protein has a possibly Q-dependent role. Finally, we report an evident relaxing constraint of the N-terminal block in Ciona and drosophilids that correlates with the absence of polyQ and which may indicate that the N-terminal portion of htt has evolved different functions in Ciona and protostomes.