TITLE: 18S and 16S rRNA genes amplicon generation for eukaryotic and prokaryotic metabarcoding
AUTHORS: Adriana  Alberti, Julie Poulain, Stefan Engelen, Karine Labadie, Sarah Romac, Isabel Ferrera, Guillaume Albini, Jean-Marc Aury, Caroline Belser, Alexis Bertrand, Corinne Cruaud, Corinne Da Silva, Carole Dossat, Frédéric Gavory, Shahinaz Gas, Julie Guy, Maud Haquelle, E'krame Jacoby, Olivier Jaillon, Arnaud Lemainque, Eric Pelletier, Gaëlle Samson, Marc Wessner, Genoscope Technical Team, Silvia G. Acinas, Marta Royo-Llonch, Francisco M. Cornejo-Castillo, Ramiro Logares, Beatriz Fernández-Gómez, Chris Bowler, Guy Cochrane, Clara Amid, Petra Ten Hoopen, Colomban De Vargas, Nigel Grimsley, Elodie Desgranges, Stefanie Kandels-Lewis, Hiroyuki Ogata, Nicole Poulton, Michael E. Sieracki, Ramunas Stepanauskas, Matthew B. Sullivan, Jennifer R. Brum, Melissa B. Duhaime, Bonnie T. Poulos, Bonnie L. Hurwitz, Stéphane Pesant, Eric Karsenti, Patrick Wincker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the 18S and 16S rRNA genes amplicon generation for eukaryotic and prokaryotic metabarcoding for the </span><span style = "font-style:italic;">Tara</span><span> Oceans expedition and is part of </span><a href="https://www.protocols.io/view/viral-to-metazoan-marine-plankton-nucleotide-seque-qv6dw9e" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Viral to metazoan marine plankton nucleotide sequences from the </span><span style = ":;font-style:italic;">Tara</span><span style = ":;"> Oceans expedition</span></a><span>. </span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/zhnfw26.jpg" /></div><div class = "text-block"><span style = "font-weight:bold;">Figure 1</span><span>: Overview of -omics analysis strategy applied on Tara Oceans samples.</span></div></div>

[STEPS]
?. Please select from the following two cases: 1. Eukaryotic 18S rRNA gene amplicon generation (Method ID: 18S_PCR)2. Prokaryotic 16S rRNA gene amplicon generation (Method ID: 16S_PCR)
?. [18S_PCR]
For generation of 18S barcodes, perform PCR amplifications with the Phusion High Fidelity PCR Master Mix with GC buffer (ThermoFisher Scientific) and the forward/reverse primer pair 1389F 5′- TTGTACACACCGCCC-3′ and 1510R 5′- CCTTCYGCAGGTTCACCTAC-3′ as described by Amaral-Zettler et al, 2009.
This primer pair yields a product of around 160 bp.
?. [18S_PCR]
Carry out PCR amplifications with the following program:initial denaturation at 98 °C for 30 s25 cycles of 10 s at 98 °C, 30 s at 57 °C, 30 s at 72 °Cfinal extension at 72 °C for 10 min
PCR is performed with a reduced number of cycles to avoid the formation of chimeras during the plateau phase of the reaction.
?. [18S_PCR]
Purify PCR products by a modified 0.6x AMPure XP beads cleanup in which after binding of DNA to the beads, the supernatant containing larger DNA fragments is kept and subsequently purified with the NucleoSpin Gel and PCR Clean-up kit.
?. [18S_PCR]
Check amplicons length and amount.Run aliquots of purified amplicons on an Agilent Bioanalyzer using the DNA High Sensitivity LabChip kit to check their lengths and quantify with a Qubit Fluorometer.
?. [16S_PCR]
For generation of 16S barcodes, perform PCR amplifications with the Phusion High Fidelity PCR Master Mix with GC buffer (ThermoFisher Scientific) and the forward/reverse primer pair 515F-Y (5′- GTGYCAGCMGCCGCGGTAA-3′) and 926R (5′- CCGYCAATTYMTTTRAGTTT-3′) 16S described by Parada et al.
This primer pair encompasses the V4 and V5 hypervariable regions, yielding a product of 400 bp.
?. [16S_PCR]
Carry out PCR amplifications with the following program:initial denaturation at 98 °C for 30 s37 cycles of 10 s at 98 °C, 30 s at 53 °C, 30 s at 72 °Cfinal extension at 72 °C for 10 min
?. [16S_PCR]
Clean up PCR products using 0.8x volumes AMPure XP beads.
?. [16S_PCR]
Check amplicons length and amount.Run aliquots of purified amplicons on an Agilent Bioanalyzer using the DNA High Sensitivity LabChip kit to check their lengths and quantify with a Qubit Fluorometer.
?. [16S_PCR]
Prepare PCR mixture containing  5 to 10 ng of total DNA template with 0.35 μM final concentration of each primer, 3% of DMSO and 1X Phusion Master Mix in 25 µl final volume.
Perform PCR  from each sample in triplicate in order to smooth the intra-sample variance and to obtain sufficient amount for Illumina sequencing.
?. [18S_PCR]
Use 100 ng of amplicons to prepare a standard Illumina library avoiding the first DNA fragmentation step.
?. [18S_PCR]
Prepare a PCR mixture containing  5 to 10 ng of total DNA template with 0.35 μM final concentration of each primer, 3% of DMSO and 1X Phusion Master Mix in 25 µl final volume.
PCR  is performed in triplicate in order to smooth the intra-sample variance and to obtain sufficient amount for Illumina sequencing.
?. [16S_PCR]
Use 100 ng of amplicons to prepare a standard Illumina library avoiding the first DNA fragmentation step.
During Illumina library preparation, a post-amplification gel size selection step is recommended to select 500-650 bp gel slices (amplicon size increased by Illumina adapters). this sizing step allows isolation of prokaryotic 16S amplicon from non -specific amplification products.