Efficient low redundancy large-scale DNA sequencing at EMBL

Voß, H.; Schwager, Christian; Wiemann, Stefan; Zimmermann, Jürgen; Stegemann, J.; Erfle, Holger; Voie, Ann Mari; Drzonek, Heiko; Ansorge, Wilhelm

doi:10.1016/0168-1656(95)00007-d

Cited by 17 publications

(10 citation statements)

References 19 publications

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“…To obtain the full length mouse sequence, we directly sequenced by primer walking 8 the BAC 13L19 (RP23 Mouse BAC Library, Research Genetics), which contains the whole mouse foxl2 locus (GenBank AI: AF522275). Human, rat, and Fugu FOXL2 sequences were assembled from GenBank (AF301906, AC105826.1, Scaffold_8165/ProtJGI_24134).…”

Section: Comparative Sequence Analysismentioning

confidence: 99%

Evolution and expression of FOXL2

Cocquet¹,

Pailhoux²,

Jaubert³

et al. 2002

Journal of Medical Genetics

261

196

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Section: Comparative Sequence Analysismentioning

confidence: 99%

Evolution and expression of FOXL2

Cocquet¹,

Pailhoux²,

Jaubert³

et al. 2002

Journal of Medical Genetics

261

196

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“…% Identity with FCV-U1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 primer technique. 17 Nucleotide and predicted amino acid sequences from the 3 clones of the FCV-U1 capsid protein gene were submitted to GenBank. j FCV-U2.…”

Section: Fcv Strainmentioning

confidence: 99%

Genetic Characterization of 2 Novel Feline Caliciviruses Isolated from Cats with Idiopathic Lower Urinary Tract Disease

Rice

Kruger

Venta

et al. 2002

Veterinary Internal Medicne

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Feline caliciviruses (FCVs) are potential etiologic agents in feline idiopathic lower urinary tract disease (I-LUTD). By means of a modified virus isolation method, we examined urine obtained from 28 male and female cats with nonobstructive I-LUTD, 12 male cats with obstructive I-LUTD, and 18 clinically healthy male and female cats. All cats had been routinely vaccinated for FCV. Two FCVs were isolated; 1 (FCV-U1) from a female cat with nonobstructive I-LUTD, and another (FCV-U2) from a male cat with obstructive I-LUTD. To determine the genetic relationship of FCV-U1 and FCV-U2 to other FCVs, capsid protein gene RNA was reverse transcribed into cDNA, amplified, and sequenced. Multiple amino acid sequence alignments and phylogenetic trees were constructed for the entire capsid protein, hypervariable region E, and the more conserved (nonhypervariable) regions A, B, D, and F. When compared to 23 other FCV isolates with known biotypes, the overall amino acid sequence identity of the capsid protein of FCV-U1 and FCV-U2 ranged from 83 to 96%; identity of hypervariable regions C and E ranged from 58 to 85%. Phylogenetically, FCV-U1 clearly separated from other FCV strains in phenograms based on nonhypervariable regions. In contrast, FCV-U2 consistently segregated with the Urbana strain in all phenograms. Clustering of isolates by geographic origin was most apparent in phenograms based on nonhypervariable regions. No clustering of isolates by biotype was apparent in any phenograms. Our results indicate that FCV-U1 and FCV-U2 are genetically distinct from other known vaccine and field strains of FCV.

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“…While end verification by Sanger sequencing can confidently verify the 5’ and 3’ ends, this method provides no information on the internal exon structure of long ORFs (unless internal primer walking [30] is carried out). We also find that the overall success rate of sequencing clones using the Sanger method is significantly lower than the 454 sequencing of amplicons.…”

Section: Resultsmentioning

confidence: 99%

Genome-wide functional annotation and structural verification of metabolic ORFeome of Chlamydomonas reinhardtii

et al. 2011

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BackgroundRecent advances in the field of metabolic engineering have been expedited by the availability of genome sequences and metabolic modelling approaches. The complete sequencing of the C. reinhardtii genome has made this unicellular alga a good candidate for metabolic engineering studies; however, the annotation of the relevant genes has not been validated and the much-needed metabolic ORFeome is currently unavailable. We describe our efforts on the functional annotation of the ORF models released by the Joint Genome Institute (JGI), prediction of their subcellular localizations, and experimental verification of their structural annotation at the genome scale.ResultsWe assigned enzymatic functions to the translated JGI ORF models of C. reinhardtii by reciprocal BLAST searches of the putative proteome against the UniProt and AraCyc enzyme databases. The best match for each translated ORF was identified and the EC numbers were transferred onto the ORF models. Enzymatic functional assignment was extended to the paralogs of the ORFs by clustering ORFs using BLASTCLUST.In total, we assigned 911 enzymatic functions, including 886 EC numbers, to 1,427 transcripts. We further annotated the enzymatic ORFs by prediction of their subcellular localization. The majority of the ORFs are predicted to be compartmentalized in the cytosol and chloroplast. We verified the structure of the metabolism-related ORF models by reverse transcription-PCR of the functionally annotated ORFs. Following amplification and cloning, we carried out 454FLX and Sanger sequencing of the ORFs. Based on alignment of the 454FLX reads to the ORF predicted sequences, we obtained more than 90% coverage for more than 80% of the ORFs. In total, 1,087 ORF models were verified by 454 and Sanger sequencing methods. We obtained expression evidence for 98% of the metabolic ORFs in the algal cells grown under constant light in the presence of acetate.ConclusionsWe functionally annotated approximately 1,400 JGI predicted metabolic ORFs that can facilitate the reconstruction and refinement of a genome-scale metabolic network. The unveiling of the metabolic potential of this organism, along with structural verification of the relevant ORFs, facilitates the selection of metabolic engineering targets with applications in bioenergy and biopharmaceuticals. The ORF clones are a resource for downstream studies.

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Efficient low redundancy large-scale DNA sequencing at EMBL

Cited by 17 publications

References 19 publications

Evolution and expression of FOXL2

Evolution and expression of FOXL2

Genetic Characterization of 2 Novel Feline Caliciviruses Isolated from Cats with Idiopathic Lower Urinary Tract Disease

Genome-wide functional annotation and structural verification of metabolic ORFeome of Chlamydomonas reinhardtii

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