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.