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A next generation semiconductor based sequencing approach for the identification of meat species in DNA mixtures.

Bertolini F, Ghionda MC, D'Alessandro E, Geraci C, Chiofalo V, Fontanesi L - PLoS ONE (2015)

Bottom Line: Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species.The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification.Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural and Food Sciences, Division of Animal Sciences, University of Bologna, Viale Fanin 46, 40127, Bologna, Italy.

ABSTRACT
The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.

No MeSH data available.


Percentage of reads obtained from libraries 3A and 3B for the different species and primer pairs combinations.
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pone.0121701.g003: Percentage of reads obtained from libraries 3A and 3B for the different species and primer pairs combinations.

Mentions: To evaluate the possibility to identify species-specific reads from amplicons derived from DNA mixtures, we first compared the number of reads obtained from library 2 (that contained products separately amplified from the mammalian and avian DNA mixtures, respectively) and libraries 3A and 3B (that contained products amplified from two replicate DNA pools, including both mammalian and avian DNA; S6 Table; Figs 2 and 3). Library 2 produced a total of 24,193 mapped reads and libraries 3A and 3B gave 26,260 and 20,320 mapped reads, respectively. Primer pairs 12S_KH and 16S_KH in libraries 2 and 3A-3B obtained mapped reads for all mammalian and avian species. The proportion of reads mapped to mammalian and avian sequences obtained from the sequencing of amplicons from DNA mixtures containing all species (Libraries 3A and 3B) was similar for both amplified regions (averaged between the two libraries: 12S_KH: mammalian = 0.82 and avian = 0.18; 16S_KH: mammalian = 0.89 and avian = 0.11). Using these two primer pairs, avian fragments were amplified less efficiently compared to mammalian fragments. Similarly, in library 2 primer pair 16S_Ki did not work efficiently on the avian DNA pool compared to the mammalian DNA pool (normalized mean across species based on 1 ng of amplified DNA concentration for each species was 21.6 ± 16.0 and 231.7 ± 232.9 reads for birds and mammals, respectively; S6 Table). This lower efficiency for the avian 16S_Ki region was confirmed by the results of mapped reads obtained from the amplification of the DNA pools containing all species (library 3) in which PCR competition prevented the amplification of avian DNA (among all birds, only one turkey read was mapped in each replicate DNA pool). Among the tested mammalian DNA, the larger number of normalized reads obtained by this primer pair in both library types, 2 and 3A-3B, was obtained for the human and cattle (3577 and 2340 in library 2 and on average 5820.5 and 3762.5 in the two libraries 3, respectively), whereas the lowest number was obtained for the horse in both cases (66.3 and 23.7). Correlation of the number of reads of the same species between the two experiments (library 2 and libraries 3) for the results of primer pair 16S_Ki was 0.99, considering both mammalian data only or mammalian + avian data, providing an indirect evaluation of the experimental repeatability.


A next generation semiconductor based sequencing approach for the identification of meat species in DNA mixtures.

Bertolini F, Ghionda MC, D'Alessandro E, Geraci C, Chiofalo V, Fontanesi L - PLoS ONE (2015)

Percentage of reads obtained from libraries 3A and 3B for the different species and primer pairs combinations.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4414512&req=5

pone.0121701.g003: Percentage of reads obtained from libraries 3A and 3B for the different species and primer pairs combinations.
Mentions: To evaluate the possibility to identify species-specific reads from amplicons derived from DNA mixtures, we first compared the number of reads obtained from library 2 (that contained products separately amplified from the mammalian and avian DNA mixtures, respectively) and libraries 3A and 3B (that contained products amplified from two replicate DNA pools, including both mammalian and avian DNA; S6 Table; Figs 2 and 3). Library 2 produced a total of 24,193 mapped reads and libraries 3A and 3B gave 26,260 and 20,320 mapped reads, respectively. Primer pairs 12S_KH and 16S_KH in libraries 2 and 3A-3B obtained mapped reads for all mammalian and avian species. The proportion of reads mapped to mammalian and avian sequences obtained from the sequencing of amplicons from DNA mixtures containing all species (Libraries 3A and 3B) was similar for both amplified regions (averaged between the two libraries: 12S_KH: mammalian = 0.82 and avian = 0.18; 16S_KH: mammalian = 0.89 and avian = 0.11). Using these two primer pairs, avian fragments were amplified less efficiently compared to mammalian fragments. Similarly, in library 2 primer pair 16S_Ki did not work efficiently on the avian DNA pool compared to the mammalian DNA pool (normalized mean across species based on 1 ng of amplified DNA concentration for each species was 21.6 ± 16.0 and 231.7 ± 232.9 reads for birds and mammals, respectively; S6 Table). This lower efficiency for the avian 16S_Ki region was confirmed by the results of mapped reads obtained from the amplification of the DNA pools containing all species (library 3) in which PCR competition prevented the amplification of avian DNA (among all birds, only one turkey read was mapped in each replicate DNA pool). Among the tested mammalian DNA, the larger number of normalized reads obtained by this primer pair in both library types, 2 and 3A-3B, was obtained for the human and cattle (3577 and 2340 in library 2 and on average 5820.5 and 3762.5 in the two libraries 3, respectively), whereas the lowest number was obtained for the horse in both cases (66.3 and 23.7). Correlation of the number of reads of the same species between the two experiments (library 2 and libraries 3) for the results of primer pair 16S_Ki was 0.99, considering both mammalian data only or mammalian + avian data, providing an indirect evaluation of the experimental repeatability.

Bottom Line: Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species.The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification.Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural and Food Sciences, Division of Animal Sciences, University of Bologna, Viale Fanin 46, 40127, Bologna, Italy.

ABSTRACT
The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.

No MeSH data available.