Journal: BMC Genomics

Article Title: Digital PCR provides sensitive and absolute calibration for high throughput sequencing

doi: 10.1186/1471-2164-10-116

Figure Lengend Snippet: A Schematic of the universal template (UT) PCR assay . The forward primer (as drawn) includes a short sequence complementary to the 8 bp dual-labeled locked nucleic acid probe on its 5' end, followed by the sequence of one of the adaptors ligated to the library molecules on its 3' end. The reverse primer (as drawn) is complementary to the sequence of the other adaptor. As the polymerase encounters the probe during strand extension, its 5' to 3' exonuclease activity cleaves the probe, releasing the fluorophore from its quencher, thus producing fluorescent signal by dequenching. B The assay is performed on a commercial microfluidic digital PCR chip. At the end of the PCR, compartments that contain amplifiable DNA molecules with sequencing adaptors properly appended give positive signal, while compartments that do not remain dark. The count of positive compartments corresponds to the number of productive library molecules in the volume loaded onto the microfluidic chip, thereby allowing measurement of the concentration of amplifiable library molecules.

Article Snippet: PCR reaction mix containing the diluted template was loaded onto Fluidigm's 12.765 Digital Array microfluidic chip.

Techniques: Sequencing, Labeling, Activity Assay, Digital PCR, Concentration Assay

Journal: Frontiers in Genetics

Article Title: Demonstration of the Use of Environmental DNA for the Non-Invasive Genotyping of a Bivalve Mollusk, the European Flat Oyster ( Ostrea edulis )

doi: 10.3389/fgene.2019.01159

Figure Lengend Snippet: Diagram (top) detailing dilutions of DNA template from different DNA extraction techniques used for SNP genotyping of environmental DNA for Ostrea edulis . The two DNA extraction methods, the Qiagen DNeasy Blood and Tissue Kit and the crude Chelex extraction, are shown at the top. DNA samples are subject to dilution step before and after the Specific Target Amplification PCR (STA PCR), here shown as a blue bar. All dilutions were with PCR grade water and samples that were not diluted are labelled ‘neat’. A scatter plot (bottom) shows the percentage of correct environmental DNA derived genotype calls in comparison to the tissue extractions for the eight dilutions. Each point represents the total result derived from a random sample of three replicates per individual oyster genotype. The black line indicates the average percentage correct eDNA genotypes across the 100 random samples. The red line indicates the average percentage correct eDNA genotypes across the 100 samples if only a single replicate is used for each individual oyster genotype.

Article Snippet: The STA product was then loaded on to a Fluidigm 96.96 Dynamic Array genotyping chip along with assays in sextuplicate, followed by a PCR at manufacturer recommended conditions and imaging on the Fluidigm EP1 data collection system.

Techniques: DNA Extraction, Extraction, Amplification, Derivative Assay, Comparison