Computational protocol: Validated methodology for quantifying infestation levels of dreissenid mussels in environmental DNA (eDNA) samples

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Protocol publication

[…] Homologous sequences of the contig sequences longer than 500 nucleotides were identified using Blast2GO software (version 2.4.2). The Non-Redundant (NR) NCBI protein database was searched using BlastX with a Cut-Off e-value of 1E-6, Cut-Off length of 30, and 20 Blast Hits. Subsequently, Gene Ontology (GO) terms were assigned according to the Gene Ontology Database with an e-value-Hit-Filter of 1E-6, annotation Cut-Off of 55, and GO Weight of 5. These results were used to predict single-copy genes using homologies with NCBI and Ensembl databases. The PCR primers for selected genes were designed using Primer3 with default parameters. [...] The dreissenid DNA from the 21 water (6 locations) samples was quantified by qPCR in three consecutive 10-fold dilutions in triplicate. To infer the effect of inhibitors in the eDNA samples, a second qPCR analysis was performed with controlled contaminations (Spikes) using the same dilutions of environmental samples but with the addition of 2 μL of 2 ng/μL zebra mussel DNA extracted from adult tissue. In all cases, the qPCR mix and thermal cycles were performed following Stage A conditions. The quantification results were analysed using 7300 SDS v1.3.1 Software (Applied Biosystems). For positive amplification samples with no inhibitors, concentrations were determined using the standard curve previously developed. The values are presented as the mean concentration (ng/L) with the corresponding standard error of the mean (SEM). A qPCR amplification was considered positive when the following conditions were met: (1) at least one of the dilutions is in the dynamic range of the standard curve and has a cycle threshold (CT) at least six cycles earlier than the no template control; (2) a proportional correspondence among decimal template dilutions and CT amplifications; (3) replicates should present a coefficient of variation lower than 0.3; and (4) specificity must be verified by a single melting peak. Finally, we calculated the estimated number of target gene copies per microliter in positive amplified samples using Avogadro constant times the ratio between DNA concentration (ng/L) and molecular weight of the PCR amplicon.The data resulting from the quantification was tested for normality by a Kolmogorov-Smirnov test, and values were transformed when necessary (square root transformation for comparisons among the seasons). The statistical analyses for concentration comparisons were performed using the mean value of the two replicates collected in each lake-season combination. These values were compared using two-tailed Student t-tests or ANOVA tests with Bonferroni post-hoc correction. All statistical analyses were computed using the IBM SPSS Statistics package (v. 20.0; IBM Corp., USA). […]

Pipeline specifications

Software tools Blast2GO, BLASTX, Primer3, SPSS
Databases NCBI Protein
Applications Miscellaneous, qPCR