PMC:5113056 / 7826-10026 JSONTXT

{"project":"2_test","denotations":[{"id":"27599472-15661851-27784062","span":{"begin":1467,"end":1469},"obj":"15661851"},{"id":"27599472-20497997-27784063","span":{"begin":1505,"end":1507},"obj":"20497997"},{"id":"27599472-20207713-27784064","span":{"begin":1509,"end":1511},"obj":"20207713"},{"id":"27599472-24451623-27784065","span":{"begin":1780,"end":1783},"obj":"24451623"},{"id":"27599472-12912839-27784066","span":{"begin":1810,"end":1812},"obj":"12912839"}],"text":"HOST GENOTYPING\nRecent re-evaluation of Cynops orientalis has resulted in the discovery of cryptic species.19, 20 To confirm the species of salamander that were part of the imported shipment, DNA was extracted from pooled organs (muscle tissue from tail, heart, tongue and stomach) of a subset of animals (n=4) using the Gentra Puregene Tissue Kit (Qiagen) according to the manufacturer's instructions. A portion of the mitochondrial NADH dehydrogenase subunit two gene (and adjacent tRNAs) was amplified with primers 3787F: 5′-TCG TGC ACC CAC TAC ACT AC-3′ and 5081R: 5′-GTC GTA GGG TCA AAG CCT GC-3′ (primer 3787F was modified slightly from Wu et al.21 to better match existing sequence data for C. orientalis). PCR amplification was conducted in 50-μL reaction volumes using GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) and 1 μL of DNA template that had been diluted 100-fold. Cycling conditions were as follow: 94 °C for 2 min; 35 cycles of 94 °C for 30 s, 52 °C for 45 s, and 72 °C for 90 s; and a final extension for 5 min at 72 °C. Amplicons were sequenced using the amplification primers and internal sequencing primer 4416F: 5′-ATA GCA TAC TCA TCC ATT GCA CA-3′.21 Chromatograms of the resulting sequences were viewed and edited (as necessary) in Lasergene 5.0 (DNASTAR, Madison, WI, USA). Final sequences were aligned with existing DNA sequences in GenBank, primarily following taxon sampling by Wu et al.19 The alignment was conducted using MAFFT22 through the online program GUIDANCE23, 24 with the following settings: 100 bootstrap repeats, 1000 maximum iterations, and ‘globalpair' pairwise alignment method. Phylogenetic analyses using maximum likelihood and Bayesian methods were conducted on the final alignment with the programs RAxML-HPC2 version 8.2.417 and MrBayes version 3.2.6,18 respectively, using the CIPRES Science Gateway.25 For both analyses, the general time reversible model with gamma distribution was used. For maximum likelihood analysis, the number of bootstrap iterations was set at 1000; all other RAxML parameters were left as default. For the Bayesian analysis, the number of generations was increased to 5 000 000 and remaining settings were default."}

{"project":"MyTest","denotations":[{"id":"27599472-15661851-27784062","span":{"begin":1467,"end":1470},"obj":"15661851"},{"id":"27599472-20497997-27784063","span":{"begin":1505,"end":1507},"obj":"20497997"},{"id":"27599472-20207713-27784064","span":{"begin":1509,"end":1512},"obj":"20207713"},{"id":"27599472-24451623-27784065","span":{"begin":1780,"end":1784},"obj":"24451623"},{"id":"27599472-12912839-27784066","span":{"begin":1810,"end":1813},"obj":"12912839"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"HOST GENOTYPING\nRecent re-evaluation of Cynops orientalis has resulted in the discovery of cryptic species.19, 20 To confirm the species of salamander that were part of the imported shipment, DNA was extracted from pooled organs (muscle tissue from tail, heart, tongue and stomach) of a subset of animals (n=4) using the Gentra Puregene Tissue Kit (Qiagen) according to the manufacturer's instructions. A portion of the mitochondrial NADH dehydrogenase subunit two gene (and adjacent tRNAs) was amplified with primers 3787F: 5′-TCG TGC ACC CAC TAC ACT AC-3′ and 5081R: 5′-GTC GTA GGG TCA AAG CCT GC-3′ (primer 3787F was modified slightly from Wu et al.21 to better match existing sequence data for C. orientalis). PCR amplification was conducted in 50-μL reaction volumes using GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) and 1 μL of DNA template that had been diluted 100-fold. Cycling conditions were as follow: 94 °C for 2 min; 35 cycles of 94 °C for 30 s, 52 °C for 45 s, and 72 °C for 90 s; and a final extension for 5 min at 72 °C. Amplicons were sequenced using the amplification primers and internal sequencing primer 4416F: 5′-ATA GCA TAC TCA TCC ATT GCA CA-3′.21 Chromatograms of the resulting sequences were viewed and edited (as necessary) in Lasergene 5.0 (DNASTAR, Madison, WI, USA). Final sequences were aligned with existing DNA sequences in GenBank, primarily following taxon sampling by Wu et al.19 The alignment was conducted using MAFFT22 through the online program GUIDANCE23, 24 with the following settings: 100 bootstrap repeats, 1000 maximum iterations, and ‘globalpair' pairwise alignment method. Phylogenetic analyses using maximum likelihood and Bayesian methods were conducted on the final alignment with the programs RAxML-HPC2 version 8.2.417 and MrBayes version 3.2.6,18 respectively, using the CIPRES Science Gateway.25 For both analyses, the general time reversible model with gamma distribution was used. For maximum likelihood analysis, the number of bootstrap iterations was set at 1000; all other RAxML parameters were left as default. For the Bayesian analysis, the number of generations was increased to 5 000 000 and remaining settings were default."}