This initial step is a single PCR amplification using tagged primers (for the identification of the sample) for 16S, 18S and ITS regions with the addition of random bases. The tagged primers allow pooling several PCR products before library preparation step, hereby reducing significantly the cost of the library preparation.

Fasteris has developed sets of PCR primers to target 3 different regions (see the list of standard Fasteris primers in the table below). However, we can adapt our protocol according to the target you need.

16S Target region

341F* (V3 region) FW primer: CCTACGGGNGGCW GCAG

805R* (V4 region) RV primer: GACTACHVGGGTATC TAATCC

Amplicon size: ~ 550 bp

Reference: Klindworth et al. 2012

(*) 16S broad range primers, 341F binds to bacteria, 805 R matching both bacteria and some archea.

ITS2 Target region

ITS3_KYO2 FW primer: GATGAAGAACGYAG YRAA

ITS4 RV primer: TCCTCCGCTTATTGAT ATGC

Amplicon Size: 300-400 bp

Reference: Toju et al. 2012

18S Target region

18S_0067a_deg FW primer: AAGCCATGCATGYCT AAGTATMA

NSR399 RV primer: TCTCAGGCTCCYTCTC CGG

Amplicon size: ~ 350 bp

Reference: Dollive et al. 2012

Fasteris | Cost-effective protocol for metagenomics study.