Nested PCR Approach for petB Gene Metabarcoding of Marine Synechococcus Populations

Abstract

The petB gene has been proposed as a high-resolution marker gene to access the diversity of marine Synechococcus populations. A systematic metabarcoding approach based on the petB gene would improve the characterization/assessment of the Synechococcus community structure in marine planktonic ecosystems.

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“…Nested PCR targeting the petB gene of Synechococcus was performed for filtered and sorted samples (Ong et al 2023). Nested PCR targeting 18S rRNA (V4 hypervariable region) was performed for sorted picoeukaryotes and nanoeukaryotes samples (Gérikas Ribeiro et al 2018).…”

“…PCR was performed using the following primer sets: Nested PCR targeting petB gene of Synechococcus for filtered and sorted samples, petB-F (5’-TACGACTGGTTCCAGGAACG-3’) and petB-634R (5’-GCTTVCGRATCATCARGAAG-3’), followed by petB-50F (5’-TYCAGGACATYGCTGAY-3’) and petB-R (5’-GAAGTGCATGAGCATGAA-3’) (Mazard et al 2012; Ong et al 2023); nested PCR targeting 18S rRNA (V4 hypervariable region) for sorted picoeukaryotes and nanoeukaryotes samples, 63F (5’-ACGCTTGTCTCAAAGATTA-3’) and 1818R (5’-ACGGAAACCTTGTTACGA-3’) (Lepere et al 2011), followed by V4F illum (5’-CCAGCASCYGCGGTAATTCC-3’) and V4R illum (5’-ACTTTCGTTCTTGATYRATGA-3’) (Piredda et al 2017); 18S rRNA seawater samples, V4F illum and V4R illum. All first and second round PCRs were performed in duplicates and pooled together.…”

“…Briefly, sequence data was processed on RStudio Version 1.4.1717 (RStudio Team 2021) using DADA2 R package Version 1.12 (Callahan et al 2016). Taxonomy was assigned against a petB reference sequence database (Farrant et al 2016) reformatted for use with DADA2 (Ong et al 2023) for Synechococcus , and PR2 database version 4.14 (Guillou et al 2013) for eukaryotes. Amplicon sequence variants (ASVs) assigned to Supergroup Opisthokonta, Division Metazoa, Fungi and Pseudofungi, class Syndiniales, as well as protists assigned as heterotrophic (based on Schneider et al 2020, full list of trophic assignment on Github) were removed and not considered in this study.…”

“…(5'-GCTTVCGRATCATCARGAAG-3'), followed by petB-50F (5'-TYCAGGACATYGCTGAY-3') and petB-R (5'-GAAGTGCATGAGCATGAA-3') (Mazard et al 2012;Ong et al 2023); nested PCR targeting 18S rRNA (V4 hypervariable region) for sorted picoeukaryotes and nanoeukaryotes samples, 63F (5'-ACGCTTGTCTCAAAGATTA-3') and 1818R (5'-ACGGAAACCTTGTTACGA-3') (Lepere et al 2011), followed by V4F illum (5'-CCAGCASCYGCGGTAATTCC-3') and V4R illum…”

“…. Taxonomy was assigned against a petB reference sequence database(Farrant et al 2016) reformatted for use with DADA2(Ong et al 2023) for Synechococcus, and PR2 database version 4.14(Guillou et al 2013) for eukaryotes. Amplicon sequence variants (ASVs) assigned to Supergroup Opisthokonta, Division Metazoa, Fungi and Pseudofungi, class…”