18S V9 of under-ice Melosira
PCR products were generated using fusion primers with the Fluidigm CS1 or CS2 universal oligomers added to their 5 ends. The MSU genomics core performed secondary PCR with dual indexed, Illumina compatible primers to complete library construction. Final PCR products were batch normalized using an Invitrogen SequalPrep DNA Normalization plate and products recovered from the plate pooled. The pool was QCd and quantified using a combination of Qubit dsDNA HS, Agilent Bioanalyzer DNA 1000 and Kapa Illumina Library Quantification qPCR assays. The pool was loaded onto two (2) standard MiSeq v2 flow cells and sequencing performed in a 2x150bp paired end format using MiSeq v2 300 cycle reagent cartridges. Custom primers for sequence reads 1 & 2 as well as index read 1 complementary to the Fluidigm CS1 and 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.