Sequence curation
Sequence curation at WormBase refers to the maintenance and annotation of the primary genomic sequence and a consensus gene set.
Genome sequence
Even though the C. elegans genome sequence is the most accurate and complete eukaryotic genome sequence, it has continually needed refinement as new evidence has been created. Many of these changes were single nucleotide insertions or deletions, however several large mis-assemblies have been uncovered. For example, in 2005 a 39 kb cosmid had to be inverted. Other improvements have come from comparing genomic DNA to cDNA sequences and analysis of RNASeq high-throughput data. When differences between the genomic sequence and transcripts are identified, re-analysis of the original genomic data often leads to modifications of the genomic sequence. The changes in the genomic sequence pose difficulties when comparing chromosomal coordinates of data derived from different releases of WormBase. There is a coordinate re-mapping program and mapping data are available to aid these comparisons. [6]
Gene structure models
All the gene-sets of the WormBase species were initially generated by gene prediction programs. Gene prediction programs give a reasonable set of gene structures, but the best of them only predict about 80% of the complete gene structures correctly. They have difficulty predicting genes with unusual structures, as well as those with a weak translation start signal, weak splice sites or single exon genes. They can incorrectly predict a coding gene model where the gene is a pseudogene and they predict the isoforms of a gene poorly, if at all.
The gene models of C. elegans, C. briggsae, C. remanei, and C. brenneri genes are manually curated. The majority of gene structure changes have been based on transcript data from large scale projects such as Yuji Kohara's EST libraries, Mark Vidal's Orfeome project (worfdb.dfci.harvard.edu/) Waterston and Hillier's Illumina data and Makedonka Mitreva's 454 data. However, other data types (e.g. protein alignments, ab initio prediction programs, trans-splice leader sites, poly-A signals and addition sites, SAGE and TEC-RED transcript tags, mass-spectroscopic peptides, and conserved protein domains) are useful in refining the structures, especially where expression is low and so transcripts are not sufficiently available. When genes are conserved between the available nematode species, comparative analysis can also be very informative.
WormBase encourages researchers to inform them via the help-desk if they have evidence for an incorrect gene structure. Any cDNA or mRNA sequence evidence for the change should be submitted to EMBL/GenBank/DDBJ; this helps in the confirmation and evidence for the gene model as WormBase routinely retrieve sequence data from these public databases. This also makes the data public, allowing appropriate reference and acknowledgement to the researchers.
When any change is made to a CDS (or Pseudogene), the old gene model is preserved as a ‘history’ object. This will have a suffix name like: “AC3.5:wp119”, where ‘AC3.5’ is the name of the CDS and the ‘119’ refers to the database release in which the change was made. The reason for the change and the evidence for the change are added to the annotation of the CDS – these can be seen in the Visible/Remark section of the CDS's ‘Tree Display’ section on the WormBase web site.
Gene nomenclature
Genes
In WormBase, a Gene is a region that is expressed or a region that has been expressed and is now a Pseudogene. Genes have unique identifiers like ‘WBGene00006415’. All C. elegans WormBase genes also have a Sequence Name, which is derived from the cosmid, fosmid or YAC clone on which they reside, for instance F38H4.7, indicating it is on the cosmid ‘F38H4’, and there are at least 6 other genes on that cosmid. If a gene produces a protein that can be classified as a member of a family, the gene may also be assigned a CGC name like tag-30 indicating that this is the 30th member of the tag gene family. Assignment of gene family names is controlled by WormBase. [7] Before publication, requests for names should be made in WormBase. [8]
There are a few exceptions to this format, like the genes cln-3.1, cln-3.2, and cln-3.3 which all are equally similar to the human gene CLN3. Gene GCG names for non-elegans species in WormBase have the 3-letter species code prepended, like Cre-acl-5, Cbr-acl-5, Cbn-acl-5.
A gene can be a Pseudogene, or can express one or more non-coding RNA genes (ncRNA) or protein-coding sequences (CDS).
Pseudogenes
Pseudogenes are genes that do not produce a reasonable, functional transcript. They may be pseudogenes of coding genes or of non-coding RNA and may be whole or fragments of a gene and may or may not express a transcript. The boundary between what is considered a reasonable coding transcript is sometimes subjective as, in the absence of other evidence, the use of weak splice sites or short exons can often produce a putative, though unsatisfactory, model of a CDS. Pseudogenes and genes with a problematic structure are constantly under review in WormBase and new evidence is used to try to resolve their status.
CDSs
Coding Sequences (CDSs) are the only part of a Gene's structure that is manually curated in WormBase. The structure of the Gene and its transcripts are derived from the structure of their CDSs.
CDSs have a Sequence Name that is derived from the same Sequence Name as their parent Gene object, so the gene ‘F38H4.7’ has a CDS called ‘F38H4.7’. The CDS specifies coding exons in the gene from the START (Methionine) codon up to (and including) the STOP codon.
Any gene can code for multiple proteins as a result of alternative splicing. These isoforms have a name that is formed from the Sequence Name of the gene with a unique letter appended. In the case of the gene bli-4 there are 6 known CDS isoforms, called K04F10.4a, K04F10.4b, K04F10.4c, K04F10.4d, K04F10.4e and K04F10.4f.
It is common to refer to isoforms in the literature using the CGC gene family name with a letter appended, for example pha-4a, however this has no meaning within the WormBase database and searches for pha-4a in WormBase will not return anything. The correct name of this isoform is either the CDS/Transcript name: F38A6.1a, or even better, the Protein name: WP:CE15998.
Gene transcripts
The transcripts of a gene in WormBase are automatically derived by mapping any available cDNA or mRNA alignments onto the CDS model. These gene transcripts will therefore often include the UTR exons surrounding the CDS. If there are no available cDNA or mRNA transcripts, then the gene transcripts will have exactly the same structure as the CDS that they are modelled on.
Gene transcripts are named after the Sequence Name of the CDS used to create them, for example, F38H4.7 or K04F10.4a.
However, if there is alternative splicing in the UTRs, which would not change the protein sequence, the alternatively spliced transcripts are named with a digit appended, for example: K04F10.4a.1 and K04F10.4a.2. If there are no isoforms of the coding gene, for example AC3.5, but there is alternative splicing in the UTRs, there will be multiple transcripts named AC3.5.1 and AC3.5.2, etc. If there are no alternate UTR transcripts the single coding_transcript is named the same as the CDS and does not have the .1 appended, as in the case of K04F10.4f.
Operons
Groups of genes which are co-transcribed as operons are curated as Operon objects. These have names like CEOP5460 and are manually curated using evidence from the SL2 trans-spliced leader sequence sites.
Non-coding RNA genes
There are several classes of non-coding RNA gene classes in WormBase:
- tRNA genes are predicted by the program ‘tRNAscan-SE’.
- rRNA genes are predicted by homology with other species.
- snRNA genes are mainly imported from Rfam.
- piRNA genes are from an analysis of the characteristic motif in these genes.
- miRNA genes have mainly been imported from miRBase. They have the primary transcript and the mature transcript marked up. The primary transcript will have a Sequence name like W09G3.10 and the mature transcript will have a letter added to this name like W09G3.10a (and if there are alternative mature transcripts, W09G3.10b, etc.).
- snoRNA genes are mainly imported from Rfam or from papers.
- ncRNA genes that have no obvious other function but which are obviously not protein-coding and are not pseudogenes are curated. Many of these have conserved homology with genes in other species. A few of these are expressed on the reverse sense to protein-coding genes.
There is also one scRNA gene.
Transposons
Transposons are not classed as genes and so do not have a parent gene object. Their structure is curated as a Transposon_CDS object with a name like C29E6.6.
Caenorhabditis elegans is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek caeno- (recent), rhabditis (rod-like) and Latin elegans (elegant). In 1900, Maupas initially named it Rhabditides elegans. Osche placed it in the subgenus Caenorhabditis in 1952, and in 1955, Dougherty raised Caenorhabditis to the status of genus.
Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene. This means the exons are joined in different combinations, leading to different (alternative) mRNA strands. Consequently, the proteins translated from alternatively spliced mRNAs usually contain differences in their amino acid sequence and, often, in their biological functions.
In computational biology, gene prediction or gene finding refers to the process of identifying the regions of genomic DNA that encode genes. This includes protein-coding genes as well as RNA genes, but may also include prediction of other functional elements such as regulatory regions. Gene finding is one of the first and most important steps in understanding the genome of a species once it has been sequenced.
Trans-splicing is a special form of RNA processing where exons from two different primary RNA transcripts are joined end to end and ligated. It is usually found in eukaryotes and mediated by the spliceosome, although some bacteria and archaea also have "half-genes" for tRNAs.
Caenorhabditis briggsae is a small nematode, closely related to Caenorhabditis elegans. The differences between the two species are subtle. The male tail in C. briggsae has a slightly different morphology from C. elegans. Other differences include changes in vulval precursor competence and the placement of the excretory duct opening. C. briggsae is frequently used to study the differences between it and the more intimately understood C. elegans, especially at the DNA and protein sequence level. Several mutant strains of C. briggsae have also been isolated that facilitate genetic analysis of this organism. C. briggsae, like C. elegans, is a hermaphrodite. The genome sequence for C. briggsae was determined in 2003.
Caenorhabditis is a genus of nematodes which live in bacteria-rich environments like compost piles, decaying dead animals and rotting fruit. The name comes from Greek: caeno- ; rhabditis = rod-like. In 1900, Maupas initially named the species Rhabditis elegans, Osche placed it in the subgenus Caenorhabditis in 1952, and in 1955, Dougherty raised Caenorhabditis to the status of genus.
UniGene was a NCBI database of the transcriptome and thus, despite the name, not primarily a database for genes. Each entry is a set of transcripts that appear to stem from the same transcription locus. Information on protein similarities, gene expression, cDNA clones, and genomic location is included with each entry.
Neurexin-2-alpha is a protein that in humans is encoded by the NRXN2 gene.
The Reference Sequence (RefSeq) database is an open access, annotated and curated collection of publicly available nucleotide sequences and their protein products. RefSeq was introduced in 2000. This database is built by National Center for Biotechnology Information (NCBI), and, unlike GenBank, provides only a single record for each natural biological molecule for major organisms ranging from viruses to bacteria to eukaryotes.
39S ribosomal protein L4, mitochondrial is a protein that in humans is encoded by the MRPL4 gene.
Trans-Spliced Exon Coupled RNA End Determination (TEC-RED) is a transcriptomic technique that, like SAGE, allows for the digital detection of messenger RNA sequences. Unlike SAGE, detection and purification of transcripts from the 5’ end of the messenger RNA require the presence of a trans-spliced leader sequence.
28S ribosomal protein S33, mitochondrial is a protein that in humans is encoded by the MRPS33 gene.
SmY ribonucleic acids are a family of small nuclear RNAs found in some species of nematode worms. They are thought to be involved in mRNA trans-splicing.
GENCODE is a scientific project in genome research and part of the ENCODE scale-up project.
sbRNA is a family of non-coding RNA first discovered in Caenorhabditis elegans. It was identified during a full transcriptome screen of the C. elegans cDNA library. Subsequent experimentation characterised sbRNA as having conserved 5' and 3' internal motifs which form a long paired stem which is interrupted with a bulge.
In molecular biology and genetics, DNA annotation or genome annotation is the process of describing the structure and function of the components of a genome, by analyzing and interpreting them in order to extract their biological significance and understand the biological processes in which they participate. Among other things, it identifies the locations of genes and all the coding regions in a genome and determines what those genes do.
The Consensus Coding Sequence (CCDS) Project is a collaborative effort to maintain a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assemblies. The CCDS project tracks identical protein annotations on the reference mouse and human genomes with a stable identifier, and ensures that they are consistently represented by the National Center for Biotechnology Information (NCBI), Ensembl, and UCSC Genome Browser. The integrity of the CCDS dataset is maintained through stringent quality assurance testing and on-going manual curation.
NeuN , a protein which is a homologue to the protein product of a sex-determining gene in Caenorhabditis elegans, is a neuronal nuclear antigen that is commonly used as a biomarker for neurons.
De novo transcriptome assembly is the de novo sequence assembly method of creating a transcriptome without the aid of a reference genome.
The G-value paradox arises from the lack of correlation between the number of protein-coding genes among eukaryotes and their relative biological complexity. The microscopic nematode Caenorhabditis elegans, for example, is composed of only a thousand cells but has about the same number of genes as a human. Researchers suggest resolution of the paradox may lie in mechanisms such as alternative splicing and complex gene regulation that make the genes of humans and other complex eukaryotes relatively more productive.
