16S amplicon sequencing of smoked fish products
The bacterial diversity was assessed in smoked and smoked-dried fishes by sequencing the V1 and V2 hypervariable regions of the 16S rRNA gene on Illumina MiSeq. The forward primer was constructed with the Illumina i7 adapter, an 8 to 10 bp barcode and primer 28F (5-GAGTTTGATCNTGGCTCAG-3). The reverse primer was constructed with the Illumina i7 adapter, an 8 to 10 bp barcode and primer 388R (5-TGCTGCCTCCCGTAGGAGT-3). Reactions were performed on ABI Veriti thermocyclers (Applied Biosytems) under the following thermal profile: 95C for 5 min, then 35 cycles of 94C for 30 sec, 54C for 40 sec, 72C for 1 min, followed by one cycle of 72C for 10 min and 4C hold. Amplification products were pooled equimolar. Each pool was size selected using Agencourt AMPure XP (BeckmanCoulter). Size selected pools were quantified using the Qubit 2.0 Fluorometer (Life Technologies) and loaded on an Illumina MiSeq 2  250 flow cell at 10 pM.
SUB6701814
Gildas SF06160919C