Annotated Protein-coding Genes Research Articles

Rice Protein Tagging Project: A Call for International Collaborations on Genome-wide In-Locus Tagging of Rice Proteins

Rice Protein Tagging Project: A Call for International Collaborations on Genome-wide In-Locus Tagging of Rice Proteins

Proteotranscriptomics assisted gene annotation and spatial proteomics of Bombyx mori BmN4 cell line

BackgroundThe process of identifying all coding regions in a genome is crucial for any study at the level of molecular biology, ranging from single-gene cloning to genome-wide measurements using RNA-seq or mass spectrometry. While satisfactory annotation has been made feasible for well-studied model organisms through great efforts of big consortia, for most systems this kind of data is either absent or not adequately precise.ResultsCombining in-depth transcriptome sequencing and high resolution mass spectrometry, we here use proteotranscriptomics to improve gene annotation of protein-coding genes in the Bombyx mori cell line BmN4 which is an increasingly used tool for the analysis of piRNA biogenesis and function. Using this approach we provide the exact coding sequence and evidence for more than 6200 genes on the protein level. Furthermore using spatial proteomics, we establish the subcellular localization of thousands of these proteins. We show that our approach outperforms current Bombyx mori annotation attempts in terms of accuracy and coverage.ConclusionsWe show that proteotranscriptomics is an efficient, cost-effective and accurate approach to improve previous annotations or generate new gene models. As this technique is based on de-novo transcriptome assembly, it provides the possibility to study any species also in the absence of genome sequence information for which proteogenomics would be impossible.

Term Matrix: a novel Gene Ontology annotation quality control system based on ontology term co-annotation patterns.

Biological processes are accomplished by the coordinated action of gene products. Gene products often participate in multiple processes, and can therefore be annotated to multiple Gene Ontology (GO) terms. Nevertheless, processes that are functionally, temporally and/or spatially distant may have few gene products in common, and co-annotation to unrelated processes probably reflects errors in literature curation, ontology structure or automated annotation pipelines. We have developed an annotation quality control workflow that uses rules based on mutually exclusive processes to detect annotation errors, based on and validated by case studies including the three we present here: fission yeast protein-coding gene annotations over time; annotations for cohesin complex subunits in human and model species; and annotations using a selected set of GO biological process terms in human and five model species. For each case study, we reviewed available GO annotations, identified pairs of biological processes which are unlikely to be correctly co-annotated to the same gene products (e.g. amino acid metabolism and cytokinesis), and traced erroneous annotations to their sources. To date we have generated 107 quality control rules, and corrected 289 manual annotations in eukaryotes and over 52 700 automatically propagated annotations across all taxa.

The Chimonanthus salicifolius genome provides insight into magnoliid evolution and flavonoid biosynthesis.

Chimonanthus salicifolius, a member of the Calycanthaceae of magnoliids, is one of the most famous medicinal plants in Eastern China. Here, we report a chromosome-level genome assembly of C.salicifolius, comprising 820.1Mb of genomic sequence with a contig N50 of 2.3Mb and containing 36651 annotated protein-coding genes. Phylogenetic analyses revealed that magnoliids were sister to the eudicots. Two rounds of ancient whole-genome duplication were inferred in the C.salicifolious genome. One is shared by Calycanthaceae after its divergence with Lauraceae, and the other is in the ancestry of Magnoliales and Laurales. Notably, long genes with > 20kb in length were much more prevalent in the magnoliid genomes compared with other angiosperms, which could be caused by the length expansion of introns inserted by transposon elements. Homologous genes within the flavonoid pathway for C.salicifolius were identified, and correlation of the gene expression and the contents of flavonoid metabolites revealed potential critical genes involved in flavonoids biosynthesis. This study not only provides an additional whole-genome sequence from the magnoliids, but also opens the door to functional genomic research and molecular breeding of C.salicifolius.

Genome Sequence of Arthrobacter sp. Phage Scuttle.

Arthrobacter phage Scuttle was isolated by enrichment from a dry soil sample (collected in Upper Darby, Pennsylvania) on host Arthrobacter sp. ATCC 21022. The genome of this phage is 43,729 bp long, has a GC content of 61.1%, and has 61 annotated protein-coding genes.

MetaEuk\u2014sensitive, high-throughput gene discovery, and annotation for large-scale eukaryotic metagenomics

BackgroundMetagenomics is revolutionizing the study of microorganisms and their involvement in biological, biomedical, and geochemical processes, allowing us to investigate by direct sequencing a tremendous diversity of organisms without the need for prior cultivation. Unicellular eukaryotes play essential roles in most microbial communities as chief predators, decomposers, phototrophs, bacterial hosts, symbionts, and parasites to plants and animals. Investigating their roles is therefore of great interest to ecology, biotechnology, human health, and evolution. However, the generally lower sequencing coverage, their more complex gene and genome architectures, and a lack of eukaryote-specific experimental and computational procedures have kept them on the sidelines of metagenomics.ResultsMetaEuk is a toolkit for high-throughput, reference-based discovery, and annotation of protein-coding genes in eukaryotic metagenomic contigs. It performs fast searches with 6-frame-translated fragments covering all possible exons and optimally combines matches into multi-exon proteins. We used a benchmark of seven diverse, annotated genomes to show that MetaEuk is highly sensitive even under conditions of low sequence similarity to the reference database. To demonstrate MetaEuk’s power to discover novel eukaryotic proteins in large-scale metagenomic data, we assembled contigs from 912 samples of the Tara Oceans project. MetaEuk predicted >12,000,000 protein-coding genes in 8 days on ten 16-core servers. Most of the discovered proteins are highly diverged from known proteins and originate from very sparsely sampled eukaryotic supergroups.ConclusionThe open-source (GPLv3) MetaEuk software (https://github.com/soedinglab/metaeuk) enables large-scale eukaryotic metagenomics through reference-based, sensitive taxonomic and functional annotation.4U3WMgJNVh8JdDg-h4V6htVideo abstract

A genome alignment of 120 mammals highlights ultraconserved element variability and placenta-associated enhancers.

BackgroundMultiple alignments of mammalian genomes have been the basis of many comparative genomic studies aiming at annotating genes, detecting regions under evolutionary constraint, and studying genome evolution. A key factor that affects the power of comparative analyses is the number of species included in a genome alignment.ResultsTo utilize the increased number of sequenced genomes and to provide an accessible resource for genomic studies, we generated a mammalian genome alignment comprising 120 species. We used this alignment and the CESAR method to provide protein-coding gene annotations for 119 non-human mammals. Furthermore, we illustrate the utility of this alignment by 2 exemplary analyses. First, we quantified how variable ultraconserved elements (UCEs) are among placental mammals. Leveraging the high taxonomic coverage in our alignment, we estimate that UCEs contain on average 4.7%–15.6% variable alignment columns. Furthermore, we show that the center regions of UCEs are generally most constrained. Second, we identified enhancer sequences that are only conserved in placental mammals. We found that these enhancers are significantly associated with placenta-related genes, suggesting that some of these enhancers may be involved in the evolution of placental mammal-specific aspects of the placenta.ConclusionThe 120-mammal alignment and all other data are available for analysis and visualization in a genome browser at https://genome-public.pks.mpg.de/and for download at https://bds.mpi-cbg.de/hillerlab/120MammalAlignment/.

A high-quality Actinidia chinensis (kiwifruit) genome

Actinidia chinensis (kiwifruit) is a perennial horticultural crop species of the Actinidiaceae family with high nutritional and economic value. Two versions of the A. chinensis genomes have been previously assembled, based mainly on relatively short reads. Here, we report an improved chromosome-level reference genome of A. chinensis (v3.0), based mainly on PacBio long reads and Hi-C data. The high-quality assembled genome is 653 Mb long, with 0.76% heterozygosity. At least 43% of the genome consists of repetitive sequences, and the most abundant long terminal repeats were further identified and account for 23.38% of our novel genome. It has clear improvements in contiguity, accuracy, and gene annotation over the two previous versions and contains 40,464 annotated protein-coding genes, of which 94.41% are functionally annotated. Moreover, further analyses of genetic collinearity revealed that the kiwifruit genome has undergone two whole-genome duplications: one affecting all Ericales families near the K-T extinction event and a recent genus-specific duplication. The reference genome presented here will be highly useful for further molecular elucidation of diverse traits and for the breeding of this horticultural crop, as well as evolutionary studies with related taxa.

A chromosomal-level genome assembly for the insect vector for Chagas disease, Triatoma rubrofasciata.

Background Triatoma rubrofasciata is a widespread pathogen vector for Chagas disease, an illness that affects approximately 7 million people worldwide. Despite its importance to human health, its evolutionary origin has not been conclusively determined. A reference genome for T. rubrofasciata is not yet available.FindingWe have sequenced the genome of a female individual with T. rubrofasciatausing a single molecular DNA sequencing technology (i.e., PacBio Sequel platform) and have successfully reconstructed a whole-genome (680-Mb) assembly that covers 90% of the nuclear genome (757 Mb). Through Hi-C analysis, we have reconstructed full-length chromosomes of this female individual that has 13 unique chromosomes (2n = 24 = 22 + X1 + X2) with a contig N50 of 2.72 Mb and a scaffold N50 of 50.7 Mb. This genome has achieved a high base-level accuracy of 99.99%. This platinum-grade genome assembly has 12,691 annotated protein-coding genes. More than 95.1% of BUSCO genes were single-copy completed, indicating a high level of completeness of the genome.ConclusionThe platinum-grade genome assembly and its annotation provide valuable information for future in-depth comparative genomics studies, including sexual determination analysis in T. rubrofasciata and the pathogenesis of Chagas disease.

Abstract 4344: Integrative functional proteogenomics for unannotated or uncharacterized proteins in cancer

Abstract Introduction: Numerous transcripts annotated as long noncoding RNAs play a central role in cancer biology. For many, their noncoding status is merely a presumption. Genome-wide sequencing of ribosomal footprints has nominated thousands of unstudied open reading frames (ORFs) within lncRNAs, representing an expansion of the proteome. Here, we investigate previously unstudied proteins in cancer cell biology. Methods: Ribosome profiling data was analyzed with RibORF. 96 hours after infection with lentivirus for selected ORFs, L1000 expression profiling was performed on 4 cell lines. A CRISPR library was screened across 8 cells lines with sgRNA sequencing on days 0, 7 and 21 post-infection. Results: We analyzed ribosomal profiling data for 14 cell lines (~320 million sequencing reads). We nominated 28530 non-canonical ORFs within annotated protein-coding genes, 6697 ORFs in annotated lncRNAs, and 1252 ORFs in pseudogenes. For further study, we selected 553 candidate ORFs that exhibited compelling features, including DNA conservation, translational efficiency, protein domain, among others. We validated protein expression for 260 of 553 ORFs (47%): 89 (16%) had supporting peptides in deep-coverage proteomics datasets; 233 (42%) expressed protein after ectopic expression of individual V5-tagged cDNAs; 10 of 30 tested untagged ORFs expressed protein by biochemical in vitro translation. Ectopic overexpression followed by RNA profiling revealed 259 cDNAs that caused cellular transcriptional changes in at least one of four cancer cell lines (A549, HA1E, A375, MCF7). 137 of the 259 (49%) were validated proteins. As controls, we generated methionine-mutant constructs: 65 of 71 mutant cDNA experiments were unable to cause similar expression changes. We used a CRISPR library to identify novel ORF dependencies in 8 Cas9-derivatized cancer cell lines (MCF7, A549, A375, PC3, HEPG2, HELA, HA1E, HT29). For 42 ORFs, ≥ 2 targeting sgRNAs produced ≤ -1 log fold depletion in ≥ 1 cell lines. These ORFs were re-tested with a second sgRNA library. Next, we investigated compelling candidates more deeply with immunoprecipitation with mass spectrometry. For the cancer outlier transcript LINC01314, which encodes a highly conserved 59 amino acid protein harboring a cortexin domain (pfam domain cl12620), we found interactions with IMMT, SAMM50, and CHCHD3, members of a mitochondrial complex. Another example is LINC00116, which encodes a highly conserved 56 amino acid protein that binds the importin-nuclear pore complex. Conclusion: We establish a framework to discover, validate, and characterize unstudied proteins. About half of tested ORFs generated a detectable protein, and of these, half impacted cellular transcription. We discover novel gene dependencies, and are elucidating mechanisms for several ORFs. Together, our work is the first large-scale attempt to study the role of unannotated proteins in cancer cell biology. Citation Format: John R. Prensner, Oana Enache, Zhe Ji, Karsten Krug, Karl R. Clauser, Xiaoping Yang, Federica Piccioni, David E. Root, Todd R. Golub. Integrative functional proteogenomics for unannotated or uncharacterized proteins in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4344.

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics.

Whole genome sequencing and genome annotation of the wild edible mushroom, Russula griseocarnosa

Russula griseocarnosa is a species of edible ectomycorrhizal fungi with medicinal properties that grows in southern China. Total DNA was isolated from a fresh fruiting body of R. griseocarnosa and subjected to sequencing using Illumina Hiseq with the PacBio RS sequencing platform. Here, we present the 64.81 Mb draft genome map of R. griseocarnosa based on 471 scaffolds and 16,128 coding protein genes. The gene annotation of protein coding genes was used to obtain corresponding annotations by blastp. Phylogenetic analysis revealed a close evolutionary relationship of R. griseocarnosa to Heterobasidion irregulare and Stereum hirsutum in the core Russulales clade. The R. griseocarnosa genome encodes a repertoire of enzymes engaged in carbohydrate and polysaccharide metabolism, along with cytochrome P450s and secondary metabolite biosynthesis. The genome content of R. griseocarnosa provides insights into the genetic basis of its reported medicinal properties and serves as a reference for comparative genomics of fungi.

LOC152742 as a biomarkerin the diagnosis of pulmonary tuberculosis infection.

Long intergenic noncoding RNAsare long noncoding transcripts from the intergenic regions of annotated protein-coding genes. The elevated expression of long noncoding RNA (lnRNA) LOC152742 has been found in tuberculosis infection yet its roles in antimycobacterial responses remain to be elucidated. In this study, the expression levels of LOC152742 in sputum, plasma of normal individuals, active tuberculosis patients, obsolete tuberculosis patients, and individuals affected with bacillus Calmette-Guerin (BCG) were analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and the sensitivity and specificity of the candidate biomarker LOC152742 were obtained. At the same time, the expression levels of LOC152742 in pulmonary epithelialand macrophages cells infected with H37Ra or H37Rv were detected by qRT-PCR. LOC152742 in sputum and plasma had a higher specificity in active tuberculosis compared with that in obsolete tuberculosis and BCG patients. Additionally, LOC152742 in pulmonary epithelial cells macrophages infected with H37Ra or H37Rv was increased significantly compared with uninfected groups indicating that LOC152742 may potentially act as a novel biomarker for diagnosis and therapy of active tuberculosis.

Genome structure and evolution of Antirrhinum majus L

Snapdragon (Antirrhinum majus L.), a member of the Plantaginaceae family, is an important model for plant genetics and molecular studies on plant growth and development, transposon biology and self-incompatibility. Here we report a near-complete genome assembly of A. majus cultivar JI7 (A. majus cv.JI7) comprising 510 Megabases (Mb) of genomic sequence and containing 37,714 annotated protein-coding genes. Scaffolds covering 97.12% of the assembled genome were anchored on eight chromosomes. Comparative and evolutionary analyses revealed that a whole-genome duplication event occurred in the Plantaginaceae around 46–49 million years ago (Ma). We also uncovered the genetic architectures associated with complex traits such as flower asymmetry and self-incompatibility, identifying a unique duplication of TCP family genes dated to around 46–49 Ma and reconstructing a near-complete ψS-locus of roughly 2 Mb. The genome sequence obtained in this study not only provides a representative genome sequenced from the Plantaginaceae but also brings the popular plant model system of Antirrhinum into the genomic age.

De novo genome assembly of the stress tolerant forest species Casuarina equisetifolia provides insight into secondary growth.

Casuarina equisetifolia (C.equisetifolia), a conifer-like angiosperm with resistance to typhoon and stress tolerance, is mainly cultivated in the coastal areas of Australasia. C.equisetifolia, making it a valuable model to study secondary growth associated genes and stress-tolerance traits. However, the genome sequence is unavailable and therefore wood-associated growth rate and stress resistance at the molecular level is largely unexplored. We therefore constructed a high-quality draft genome sequence of C.equisetifolia by a combination of Illumina second-generation sequencing reads and Pacific Biosciences single-molecule real-time (SMRT) long reads to advance the investigation of this species. Here, we report the genome assembly, which contains approximately 300 megabases (Mb) and scaffold size of N50 is 1.06Mb. Additionally, gene annotation, assisted by a combination of prediction and RNA-seq data, generated 29827 annotated protein-coding genes and 1983 non-coding genes, respectively. Furthermore, we found that the total number of repetitive sequences account for one-third of the genome assembly. Here we also construct the genome-wide map of DNA modification, such as two novel forms N6 -adenine (6mA) and N4-methylcytosine (4mC) at the level of single-nucleotide resolution using single-molecule real-time (SMRT) sequencing. Interestingly, we found that 17% of 6mA modification genes and 15% of 4mC modification genes also included alternative splicing events. Finally, we investigated cellulose, hemicellulose, and lignin-related genes, which were associated with secondary growth and contained different DNA modifications. The high-quality genome sequence and annotation of C.equisetifolia in this study provide a valuable resource to strengthen our understanding of the diverse traits of trees.

Characterization of the complete mitochondrial genome of Drechslerella brochopaga, a fungal species trapping nematodes with constricting rings

We assembled and annotated the complete mitochondrial genome of Drechslerella brochopaga. This mitogenome is a closed circular molecule of 193,195 bp in length with a GC content of 26%, including 14 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA) genes. We analyzed the phylogenetic relationship between D. brochopaga and other 26 ascomycetes using the 14 annotated protein-coding genes of the mitochondrial genomes.

SpinachBase: a central portal for spinach genomics.

Spinach (Spinacia oleracea L.) is a nutritious vegetable enriched with many essential minerals and vitamins. A reference spinach genome has been recently released, and additional spinach genomic resources are being rapidly developed. Therefore, there is an urgent need of a central database to store, query, analyze and integrate various resources of spinach genomic data. To this end, we developed SpinachBase (http://spinachbase.org), which provides centralized public accesses to genomic data as well as analytical tools to assist research and breeding in spinach. The database currently stores the spinach reference genome sequence, and sequences and comprehensive functional annotations of protein-coding genes predicted from the genome. The database also contains gene expression profiles derived from RNA-Seq experiments as well as highly co-expressed genes and genetic variants called from transcriptome sequences of 120 cultivated and wild Spinacia accessions. Biochemical pathways have been predicted from spinach protein-coding genes and are available through a pathway database (SpinachCyc) within SpinachBase. SpinachBase provides a suite of analysis and visualization tools including a genome browser, sequence similarity searches with BLAST, functional enrichment and functional classification analyses and functions to query and retrieve gene sequences and annotations.

Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani.

The 90-kDa heat shock protein (HSP90) of eukaryotes is a highly abundant and essential chaperone required for the maturation of regulatory and signal proteins. In the protozoan parasite Leishmania donovani, causative agent of the fatal visceral leishmaniasis, HSP90 activity is essential for cell proliferation and survival. Even more importantly, its inhibition causes life cycle progression from the insect stage to the pathogenic, mammalian stage. To unravel the molecular impact of HSP90 activity on the parasites' gene expression, we performed a ribosome profiling analysis of L. donovani, comparing genome-wide protein synthesis patterns in the presence and absence of the HSP90-specific inhibitor radicicol and an ectopically expressed radicicol-resistant HSP90 variant. We find that ribosome-protected RNA faithfully maps open reading frames and represents 97% of the annotated protein-coding genes of L. donovani. Protein synthesis was found to correlate poorly with RNA steady-state levels, indicating a regulated translation as primary mechanism for HSP90-dependent gene expression. The results confirm inhibitory effects of HSP90 on the synthesis of Leishmania proteins that are associated with the pathogenic, intracellular stage of the parasite. Those include heat shock proteins, redox enzymes, virulence-enhancing surface proteins, proteolytic pathways, and a complete set of histones. Conversely, HSP90 promotes fatty acid synthesis enzymes. Complementing radicicol treatment with the radicicol-resistant HSP90rr variant revealed important off-target radicicol effects that control a large number of the above-listed proteins. Leishmania lacks gene-specific transcription regulation and relies on regulated translation instead. Our ribosome footprinting analysis demonstrates a controlling function of HSP90 in stage-specific protein synthesis but also significant, HSP90-independent effects of the inhibitor radicicol. IMPORTANCE Leishmania parasites cause severe illness in humans and animals. They exist in two developmental stages, insect form and mammalian form, which differ in shape and gene expression. By mapping and quantifying RNA fragments protected by protein synthesis complexes, we determined the rates of protein synthesis for >90% of all Leishmania proteins in response to the inhibition of a key regulatory protein, the 90-kDa heat shock protein. We find that Leishmania depends on a regulation of protein synthesis for controlling its gene expression and that heat shock protein 90 inhibition can trigger the developmental program from insect form to mammalian form of the pathogen.

Combining RNA-seq data and homology-based gene prediction for plants, animals and fungi

BackgroundGenome annotation is of key importance in many research questions. The identification of protein-coding genes is often based on transcriptome sequencing data, ab-initio or homology-based prediction. Recently, it was demonstrated that intron position conservation improves homology-based gene prediction, and that experimental data improves ab-initio gene prediction.ResultsHere, we present an extension of the gene prediction program GeMoMa that utilizes amino acid sequence conservation, intron position conservation and optionally RNA-seq data for homology-based gene prediction. We show on published benchmark data for plants, animals and fungi that GeMoMa performs better than the gene prediction programs BRAKER1, MAKER2, and CodingQuarry, and purely RNA-seq-based pipelines for transcript identification. In addition, we demonstrate that using multiple reference organisms may help to further improve the performance of GeMoMa. Finally, we apply GeMoMa to four nematode species and to the recently published barley reference genome indicating that current annotations of protein-coding genes may be refined using GeMoMa predictions.ConclusionsGeMoMa might be of great utility for annotating newly sequenced genomes but also for finding homologs of a specific gene or gene family. GeMoMa has been published under GNU GPL3 and is freely available at http://www.jstacs.de/index.php/GeMoMa.

Consistent prediction of GO protein localization

The GO-Cellular Component (GO-CC) ontology provides a controlled vocabulary for the consistent description of the subcellular compartments or macromolecular complexes where proteins may act. Current machine learning-based methods used for the automated GO-CC annotation of proteins suffer from the inconsistency of individual GO-CC term predictions. Here, we present FGGA-CC+, a class of hierarchical graph-based classifiers for the consistent GO-CC annotation of protein coding genes at the subcellular compartment or macromolecular complex levels. Aiming to boost the accuracy of GO-CC predictions, we make use of the protein localization knowledge in the GO-Biological Process (GO-BP) annotations to boost the accuracy of GO-CC prediction. As a result, FGGA-CC+ classifiers are built from annotation data in both the GO-CC and GO-BP ontologies. Due to their graph-based design, FGGA-CC+ classifiers are fully interpretable and their predictions amenable to expert analysis. Promising results on protein annotation data from five model organisms were obtained. Additionally, successful validation results in the annotation of a challenging subset of tandem duplicated genes in the tomato non-model organism were accomplished. Overall, these results suggest that FGGA-CC+ classifiers can indeed be useful for satisfying the huge demand of GO-CC annotation arising from ubiquitous high throughout sequencing and proteomic projects.