An integrated computational pipeline and database to support whole-genome sequence annotation

Mungall, CJ; Misra, Sima; Berman, Benjamin P.; Carlson, Joseph W.; Frise, Erwin; Harris, Nomi L.; Marshall, Brad; Shu, Shengqiang; Kaminker, JS; Prochnik, SE; Smith, CD; Smith, E. J.; Tupy, JL; Wiel, C. van der; Rubin, GM; Lewis, Stephen E.

doi:10.1186/gb-2002-3-12-research0081

Cited by 46 publications

(12 citation statements)

References 35 publications

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“…Our work fulfills the demand for a unified approach to TE annotation that capitalizes on the strength of multiple TE detection methods [3] and places TE annotation on common conceptual framework with gene annotation [5–9]. Compared with annotations generated for the Release 3 sequence [18], we confirmed precisely 743 out of 1,572 TE annotations.…”

Section: Discussionsupporting

confidence: 56%

Combined Evidence Annotation of Transposable Elements in Genome Sequences

et al. 2005

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Transposable elements (TEs) are mobile, repetitive sequences that make up significant fractions of metazoan genomes. Despite their near ubiquity and importance in genome and chromosome biology, most efforts to annotate TEs in genome sequences rely on the results of a single computational program, RepeatMasker. In contrast, recent advances in gene annotation indicate that high-quality gene models can be produced from combining multiple independent sources of computational evidence. To elevate the quality of TE annotations to a level comparable to that of gene models, we have developed a combined evidence-model TE annotation pipeline, analogous to systems used for gene annotation, by integrating results from multiple homology-based and de novo TE identification methods. As proof of principle, we have annotated “TE models” in Drosophila melanogaster Release 4 genomic sequences using the combined computational evidence derived from RepeatMasker, BLASTER, TBLASTX, all-by-all BLASTN, RECON, TE-HMM and the previous Release 3.1 annotation. Our system is designed for use with the Apollo genome annotation tool, allowing automatic results to be curated manually to produce reliable annotations. The euchromatic TE fraction of D. melanogaster is now estimated at 5.3% (cf. 3.86% in Release 3.1), and we found a substantially higher number of TEs (n = 6,013) than previously identified (n = 1,572). Most of the new TEs derive from small fragments of a few hundred nucleotides long and highly abundant families not previously annotated (e.g., INE-1). We also estimated that 518 TE copies (8.6%) are inserted into at least one other TE, forming a nest of elements. The pipeline allows rapid and thorough annotation of even the most complex TE models, including highly deleted and/or nested elements such as those often found in heterochromatic sequences. Our pipeline can be easily adapted to other genome sequences, such as those of the D. melanogaster heterochromatin or other species in the genus Drosophila.

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Section: Discussionsupporting

confidence: 56%

Combined Evidence Annotation of Transposable Elements in Genome Sequences

et al. 2005

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“…Although the ORFs encoded by our candidate ncRNAs are indeed short and their translated sequences have no similarity to known proteins (BLASTX analysis of these sequences was conducted as part of the D. melanogaster 3.1 annotation pipeline) (12,15), the possibility remains that some of these sequences encode novel small peptides. To assess this possibility, we asked whether sequence conservation in the D. pseudoobscura genome is greater within the ORF than in the remainder of the transcript.…”

Section: Resultsmentioning

confidence: 99%

“…ncRNA genes, we screened for cDNAs that did not intersect existing annotations (12,15). Additional analyses (transcript length, ORF length and composition, initiating codon, polyadenylation length and consensus sites, genomic extent of transcription unit, and splice site prediction) were accomplished by using PERL scripts and the WU-BLAST 2.0 (http:͞͞blast.wustl.edu) and SIM4 (16) …”

Section: Methodsmentioning

confidence: 99%

Identification of putative noncoding polyadenylated transcripts in Drosophila melanogaster

Tupy

Bailey

Dailey

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

109

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authors note errors in three sentences in the second paragraph on page 15042, left column. The corrected paragraph is reprinted below, and the revised sentences appear in boldface. The authors are grateful to Dr. Benny Abraham for identifying the errors.The reported SNP for CD24 is a replacement of C at nucleotide 226 by T (C3T) in the coding region of exon 2 (GenBank accession no. NM013230), which results in a substitution of Ala at amino acid 57 by Val near the GPI anchorage site of the mature protein. The genomic DNA was isolated from Ϸ5 ϫ 10 6 human peripheral blood leukocytes (PBL) by using the QIAamp DNA Blood Minikit (Qiagen, Valencia, CA). DNA fragments bearing this SNP site were amplified by PCR by using a forward primer (TTG TTG CCA CTT GGC ATT TTT GAG GC) and a reverse primer (GGA TTG GGT TTA GAA GAT GGG GAA A). The PCR conditions were as follows: 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, for 35 cycles. The predicted CD24 PCR fragment is 453 bp long. The C3T change yielded a BstXI restriction enzyme site at nucleotide 225, which allowed us to differentiate these two different CD24 alleles by restriction fragment length polymorphism analysis. Briefly, an aliquot of CD24 PCR products was digested with BstXI for 16 h at 50°C. The digested products were then separated in a 2.5% agarose gel. The predicted digestion pattern is as follows: PCR products of T226 allele will be cut into two small fragments (325 and 129 bp), whereas those of the C226 will be completely resistant. A combination of the two types of the products at close to 50% levels indicates the heterozygosity of the subject. 1073͞pnas.0501422102), the authors note the following regarding the homology of conceptual translations of putative noncoding RNA (pncr) transcripts to known proteins. The report correctly states that for all candidate noncoding transcripts curated in this study, BLASTX analyses using default parameters return no results. However, subsequent analyses of those candidates designated by this study as pncr genes using BLASTP with a PAM30 substitution matrix has revealed homology to known proteins for 2 of the 17 genes listed in Table 2: pncr005:2R and pncr006:X. Homology to a conceptual translation was found for a third transcript, pncr007:3R. We are therefore withdrawing the pncr gene designations in these three cases. No protein homology is detected for other pncr transcripts under these parameters.

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“…For other low-quality reads, we used PHRED to call bases and score quality. RT-PCR and oligo sequences were aligned to the genomic sequence by using SIM4WRAP (11). Matches were filtered by using the BERKELEY OUTPUT PARSER (11).…”

Section: Methodsmentioning

confidence: 99%

“…RT-PCR and oligo sequences were aligned to the genomic sequence by using SIM4WRAP (11). Matches were filtered by using the BERKELEY OUTPUT PARSER (11). The control, GENSCAN, FGENESH, and Heidelberg (2) predictions and associated oligos were loaded into a modified release 3.1 gadfly database, and each prediction was visualized with aligned RT-PCR products and oligos by using the APOLLO genome annotation browser and editor (15).…”

Section: Methodsmentioning

confidence: 99%

A computational and experimental approach to validating annotations and gene predictions in theDrosophila melanogastergenome

Yandell

Bailey

Misra

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Five years after the completion of the sequence of the Drosophila melanogaster genome, the number of protein-coding genes it contains remains a matter of debate; the number of computational gene predictions greatly exceeds the number of validated gene annotations. We have assembled a collection of >10,000 gene predictions that do not overlap existing gene annotations and have developed a process for their validation that allows us to efficiently prioritize and experimentally validate predictions from various sources by sequencing RT-PCR products to confirm gene structures. Our data provide experimental evidence for 122 proteincoding genes. Our analyses suggest that the entire collection of predictions contains only Ϸ700 additional protein-coding genes. Although we cannot rule out the discovery of genes with unusual features that make them refractory to existing methods, our results suggest that the D. melanogaster genome contains Ϸ14,000 protein-coding genes.gene number ͉ validation ͉ genome annotation T he total number of protein-coding genes in the Drosophila melanogaster genome remains a subject of debate. Whereas those who curated the D. melanogaster genome concluded that the annotated 13,659 genes in the 3.1 release likely constitute 95% of all protein-coding genes (1), others researchers have concluded that many, possibly thousands, of protein-coding genes remain unannotated (2). Two issues have fueled the debate surrounding gene number in D. melanogaster: the large numbers of computational gene predictions located within intergenic regions and varying standards of experimental evidence for concluding that a gene prediction corresponds to a real gene.As of release 3.1, Ϸ50% of the D. melanogaster genome is intergenic. Running the gene prediction program GENSCAN (3) on every intergenic region in the D. melanogaster genome results in 10,644 gene predictions spread amongst 62 megabases (Mb) of annotation-free sequence. Surely some of these predictions are real, but how many? The best way to answer this question is to subject a representative sample of the gene predictions to some validation procedure.The design and interpretation of experiments intended to assay expression of genes that have been predicted computationally have become controversial. One approach is to rely on hybridization to microarrays or RT-PCR assays for transcript expression (2), with the detection of a product by agarose gel electrophoresis taken as confirmation of the corresponding gene prediction. However, as our results show, unless the diagnostic PCR product includes a splice junction, amplification of residual genomic DNA and detection of unprocessed transcripts may lead to false verifications of gene predictions. It has also been critical to determine the sequence, and not just the size, of the PCR products (4).One way to obtain spliced cDNAs for sequencing is to perform RT-PCR with a 3Ј-oligo(dT) primer and an upstream PCR primer located in the prediction's 5Ј-most exon. The advantages of this approach are that it requires only a ...

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An integrated computational pipeline and database to support whole-genome sequence annotation

Cited by 46 publications

References 35 publications

Combined Evidence Annotation of Transposable Elements in Genome Sequences

Combined Evidence Annotation of Transposable Elements in Genome Sequences

Identification of putative noncoding polyadenylated transcripts in Drosophila melanogaster

A computational and experimental approach to validating annotations and gene predictions in theDrosophila melanogastergenome

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