16S barcoding strawberry
Metagenomic sequencing was performed on an Illumina Novaseq platform (paired end 150 bp reads) by Biosquence (custom design assay, Quito, EC), following a comprehensive workflow which combines a benchtop sequencing system, onboard primary analysis, and secondary analysis using MiSeq Reporter or BaseSpace (Illumina, USA). The 16S rRNA V3-V4 region was amplified with the bacterial primers 341F (5-CCTACGGNGGCWGCAG-3) and 805R (5-GACTACHVGGGTATCTAATCC-3) (Klindworth et al., 2013), added Illumina sequencing adapters and dualindex barcodes to the amplicon target. All PCR reactions involved the KAPA HiFi HotStart ReadyMix (Sigma Aldrich, USA). A washing step using magnetic beads was applied to purify the 16S, V3, and V4 amplicons from free primers and primer dimer species. Using the full complement of Nextera XT indices, up to 96 libraries can be pooled together for sequencing. Sequence of MiSeqUsing paired 300bp reads, and MiSeq v3 reagents, the ends of each read are overlapped to generate highquality, fulllength reads of the V3 and V4 region. Library preparation consisted of adding indices to each end of the previously obtained amplicons. Indices are unique sequences that are assigned to all amplicons in the same sample to distinguish them from amplicons in other samples, allowing multiple samples to be sequenced in parallel and independent data for each (Illumina workflow). The second wash is performed using magnetic beads to clean the final library. Finally, the purified libraries are quantified and qualified to determine their suitability for sequencing.