Fig. 1
From: Comprehensive antibiotic-linked mutation assessment by resistance mutation sequencing (RM-seq)
RM-seq workflow. a Schematic view of the experimental design. A large population of resistant clones are selected in vitro from multiple independent cultures. The mutation repertoire selected in a resistance-associated locus is then identified by amplicon deep sequencing. Analysis of the differential abundance of resistance mutations among a resistant clone library before and after a subsequent in vitro (cross-resistance) or in vivo (mouse infection model) selection pressure permits the screening of pleiotropic resistance mutations. b Amplicon library preparation and deep sequencing. Unique molecular barcodes are introduced by linear PCR (template elongation) using a primer comprising a 16Â bp random sequence (green, yellow and blue part of the middle section of the linear PCR primer). Nested exponential PCR using three primers adds Illumina adapters (blue and yellow primer tails) and indices for multiplexing (black and grey primer sections). Grouping of the reads sharing identical 16Â bp barcodes allows differentiation of true SNPs (red, pink and yellow) from sequencing errors (black) by consensus sequence reconstruction using multiple reads from the initial template molecule. Counting the number of unique barcodes for each variant provides an unbiased relative quantification of sequence variants. c Bioinformatics analysis pipeline. The diagram represents the different steps in the data processing pipeline. The bioinformatics programs used in the pipeline are indicated in italics