Temporal identification of the appearance and fixation of mutations

The time of acquisition and fixation of the nonsynonymous SNP present in locus aacA (encoding the acetyl-CoA carboxylase α subunit) was identified by selecting 10 colonies of NCTC8325s (NCTC8325 vWbp_EcoRI) during every week after the coinoculation with NCTC8325 and NCTC8325s and performing sequencing of the PCR products obtained with primers SNP_aacA_1m to SNP_aacA_2c. To identify the time of fixation of the SNP present in 3% of the population, we extracted DNA from 400 colonies isolated from milk samples at days 14, 22, and 29 and performed PCRs to obtain an amplicon of pool DNA with primers 3SNP_hp_1m to 3SNP_hp_2c. These primers contained overhang adapter sequences that were necessary for subsequent sequencing by Illumina (MiSeq Nano 2 × 250 bp). Mapping to the reference genome permitted to identify the counts present in nucleotide 401 corresponding to SNP present in 3% of the population.

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