Parmelia sulcata
We submitted a set of 1 PCR amplification products of the ITS2 region to the Genomics Core at Michigan State University. These PCR products were generated with primers FDGITS2-f and FDGITS2-r which included Fluidigm CS1 or CS2 universal oligomer sequences at their 5 ends. The MSU Genomics Core completed library preparation by secondary PCR targeting the Fluidigm CS1/CS2 oligos at the ends of our primary PCR products. Primers used for 2 PCR included dual indexed, Illumina library compatible sequences. Finished libraries were bulk normalized using Invitrogen SequalPrep DNA Normalization Plates and the recovered products pooled. The pool was QCd and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000 and Kapa Illumina Library Quantification qPCR assays. This pool was loaded onto an Illumina MiSeq v2 Standard flow cell and sequencing performed in a 2x250bp paired end format using a MiSeq v2 500 cycle reagent cartridge. Custom sequencing and index primers complementary to the Fluidigm CS1/CS2 oligos were added to appropriate wells of the reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.