Seawater COI metabarcoding site 3 SMJW replicate 3
A 313 bp region of the cytochrome c oxidase subunit 1 (COI) gene was amplified in triplicate from each DNA sample using the forward mlCOIint and reverse jgHCO2198 universal primers from Leray 2013 containing Illumina sequencing adapters. Each PCR reaction consisted of 1 mL of 10mM of each forward and reverse primer, 12.5 mL of KAPA HiFi HotStart DNA Polymerase (HotStart and Ready Mix formulation, KAPA Biosystems), 8.5 mL of RNA free water, and 2 mL of genomic DNA. A unique combination of Illumina dual indexes (Nextera XT Index Kit) was attached to the Illumina sequencing adapters of each pooled sample in a second PCR. DNA libraries were loaded into the Illumina MiSeq sequencing platform (v3 chemistry) at KAUST (Bioscience CORE Labs). Twenty percent phiX was included for internal control. Sequence data were automatically demultiplexed using MiSeq Reporter (v2), and forward and reverse reads were assigned to samples.