The resistome profile of California retail milk before and after RT incubation. a Within the sequencing data from HiSeq 4000, ARGs were undetectable prior to RT incubation in both HTST (n = 5) and raw (n = 6) milk samples. The prevalence of ARGs significantly increased in all RT-incubated raw milk (n = 7), with a single HTST sample (out of 6) possessing measurable ARGs. A Wilcoxon rank sum test was used for statistical comparison of the abundance of ARGs between HTST and raw milks (#P < 0.05). Boxplots denote the interquartile (IQR) between the first and third quartiles (25th and 75th percentiles, respectively) and median is denoted by the horizontal line. b RT incubation enriched the ARGs conferring resistance to 4 classes of antibiotics. A balanced design of raw milk samples before and after RT incubation from both HiSeq 4000 (n = 13) and NovaSeq S4 (n = 8) were included in this analysis. A Wilcoxon signed-rank test was used to assess the significant changes of ARG abundance belonging to each class of antibiotics, and an FDR correction was applied for multiple comparisons (★P < 0.05). c The observed ARGs in raw milk (n = 21) were predicted originate from 9 different bacterial families. Edge thickness indicates the normalized abundance of ARGs (rounded squares; colored by class of antibiotics) from a predicted bacterial family (circles). Node size represents the number of connections (degree). Detailed information of ARGs and bacteria networks is available in Supplementary Data 2.