Fig. 2

Millstone accurately detects genomic variants and can iteratively version genomes. a Millstone was used to analyze genomic clones involved in generating and rationally optimizing a genomically recoded organism. MAGE [2] and CAGE [3] were used to generate the C321. ΔA strain of Escherichia coli [7]. With sequencing data from these strains, Millstone confirmed the designed mutations, identified and annotated off-target mutations, and generated a new reference genome. Further reversion of variants was performed with MAGE to improve the strain’s fitness [8], and a final reference genome was generated. b Analysis of 11 escapee clones from a biocontainment selection with a synthetic non-standard amino acid (nsAA) auxotrophy [9] identified two escape mechanisms, either mutation of tyrS or disruption of lon. c Millstone can also be used for adaptive laboratory evolution studies. We employed Millstone to analyze mutations across 115 clones in the Tenaillon et al. [10] high-temperature evolution experiment. Millstone was used to create a new reference genome for the ancestral strain from REL606, the closest available reference genome, and called variants against this new reference. Millstone reports 99.2% of SNVs, deletions, and mobile elements found by the Tenaillon pipeline, as well some not identified in the original study (Additional file 1: Table S2). GRO genomically recoded organism, Ref. reference, SNV single-nucleotide variant