Library preparation and sequencing
rbc L libraries preparation and sequencing were carried out by Sistemas Genómicos S. L. (Paterna, Valencia, Spain). Purified DNA from the six PCR replicates was pooled in one sample and then quantified with Qubit 3.0 (Invitrogen, Life Technologies, Grand Island, New York, US). 5 ng of pooled PCR products from each sample were subjected to indexing PCR on 50 µL of reaction mixture containing KAPA HiFi Hot Start DNA Polymerase (Kapa Biosystems, Wilmington, Delaware, US) and a specific primers combination to enable multiplexing of all PCR products in the same sequencing run. PCR products were purified with Agencourt AMPure XP beads (Beckman-Coulter, Brea, California, US). Then, quality and quantity of purified amplicons was assessed using a 4200 TapeStation (Agilent Technologies, Santa Clara, California, US) with a High Sensitivity D1000 ScreenTape. Finally, rbc L libraries were pooled and paired-end sequenced (2x250 bp) on MiSeq 2500 instrument (Illumina, San Diego, California, US) using a Micro MiSeq Reagent Kit v2.