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BMC Genomics
2020 May 11;211:352. doi: 10.1186/s12864-020-6762-2.
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Assessment of databases to determine the validity of β- and γ-carbonic anhydrase sequences from vertebrates.
Zolfaghari Emameh R
,
Kuuslahti M
,
Nosrati H
,
Lohi H
,
Parkkila S
.
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BACKGROUND: The inaccuracy of DNA sequence data is becoming a serious problem, as the amount of molecular data is multiplying rapidly and expectations are high for big data to revolutionize life sciences and health care. In this study, we investigated the accuracy of DNA sequence data from commonly used databases using carbonic anhydrase (CA) gene sequences as generic targets. CAs are ancient metalloenzymes that are present in all unicellular and multicellular living organisms. Among the eight distinct families of CAs, including α, β, γ, δ, ζ, η, θ, and ι, only α-CAs have been reported in vertebrates.
RESULTS: By an in silico analysis performed on the NCBI and Ensembl databases, we identified several β- and γ-CA sequences in vertebrates, including Homo sapiens, Mus musculus, Felis catus, Lipotes vexillifer, Pantholops hodgsonii, Hippocampus comes, Hucho hucho, Oncorhynchus tshawytscha, Xenopus tropicalis, and Rhinolophus sinicus. Polymerase chain reaction (PCR) analysis of genomic DNA persistently failed to amplify positive β- or γ-CA gene sequences when Mus musculus and Felis catus DNA samples were used as templates. Further BLAST homology searches of the database-derived "vertebrate" β- and γ-CA sequences revealed that the identified sequences were presumably derived from gut microbiota, environmental microbiomes, or grassland ecosystems.
CONCLUSIONS: Our results highlight the need for more accurate and fast curation systems for DNA databases. The mined data must be carefully reconciled with our best knowledge of sequences to improve the accuracy of DNA data for publication.
Fig. 1. Predicted genomic location of (a) a β-CA gene in Mus musculus, strain NOD/ShiLtJ (scaffold LVXS01065484.1: 870–1430) and (b) a γ-CA gene in Xenopus tropicalis (scaffold GL180697.1: 4765-5075)
Fig. 2. Multiple sequence alignment (MSA) of β-CA protein sequences from vertebrates. The highly conserved amino acids are shown by highlighted vertical bands
Fig. 3. Multiple sequence alignment (MSA) of γ-CA protein sequences from vertebrates. The highly conserved amino acids are shown by highlighted vertical bands
Fig. 4. PCR analysis of the γ-CA gene from F. catus and β-CA gene from M. musculus. Samples from two animals of both species were included in the analysis, and primer pairs P1, P3, P5, and P8 were selected based on preliminary experiments. a shows the results from the first round of PCR. The bands nearest to the estimated correct size (red arrows) are marked with red circles (1–9). These bands were isolated, and the purified DNAs were used as templates for the second round of PCR. The results are shown in b. The amplified products from samples 3, 4, 8, and 9 were subsequently subjected to DNA sequencing
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