Primer design and optimisation

Forward and reverse oligonucleotide primers were designed with Primer-Blast at the National Centre for Biotechnology Information (NCBI) [103] to amplify each of the 34 C. gigas NRs. Primers were 18–23 nt, with a GC content of 40–60 % and produced predicted amplicons of 100–200 bp (Additional file 3). The primer pairs were optimized by changing final primer concentration, temperature and/or final MgCl₂ concentration to reach a primer pair efficiency between 90 and 115 %. The efficiency was tested by a dilution series resulting in a standard curve with a slope between 3.0 and 3.55. The efficiency was calculated as follows [104]: Efficiency (E) = 10^(-1/slope).

Each primer pair amplification product was verified by sequencing, using a common polymerase chain reaction (PCR) with the GoTaq system (Promega) for amplification and the products were purified with the QIAquick PCR Purification Kit (Qiagen, UK). Sequencing was conducted by Eurofins MWG Operon (Ebersberg, Germany).