A pan-genome method to determine core regions of the Bacillus subtilis and Escherichia coli genomes [version 2; peer review: 2 approved]

Background: Synthetic engineering of bacteria to produce industrial products is a burgeoning field of research and application. In order to optimize genome design, designers need to understand which genes are essential, which are optimal for growth, and locations in the genome that will be tolerated by the organism when inserting engineered cassettes. Methods: We present a pan-genome based method for the identification of core regions in a genome that are strongly conserved at the species level. Results: We show that the core regions determined by our method contain all or almost all essential genes. This demonstrates the accuracy of our method as essential genes should be core genes. We show that we outperform previous methods by this measure. We also explain why there are exceptions to this rule for our method. Conclusions: We assert that synthetic engineers should avoid deleting or inserting into these core regions unless they understand and are manipulating the function of the genes in that region. Similarly, if the designer wishes to streamline the genome, non-core regions and in particular low penetrance genes would be good targets for deletion. Care should be taken to remove entire cassettes with similar penetrance of the genes within cassettes as they may harbor toxin/antitoxin genes which need to be removed in tandem. The bioinformatic approach introduced here saves considerable time and effort relative to knockout studies on single isolates of a given species and captures a broad understanding of the conservation of genes that are core to a species. (such as toxin/anti-toxin gene pairs) and more distant orthologs not captured in OGCs.We which might be undergoing gene gene regions tend of transferred This extensive, complex report provides details about a new methodological approach for the detection of ‘core’ regions of Bacillus subtilis and Escherichia coli . Core regions are defined within the pan-genome context of conserved genomic loci for the two bacterial species, as a case study. An underlying assumption and implicit goal of the study is that the detected core regions largely correspond to ‘essential’ genes, as those have been determined by independent experimental methodology, with implications for synthetic engineering of bacteria. For both purposes, namely the detection of core regions and the correspondence of those to essential genes, this report is an important contribution, especially as it resolves the connection of core to essential genes. It brings to the forefront the use of pangenome analysis for synthetic biology – a factor that so far has been, to our amazement (!), ignored by biotechnologists. Solid work and a significant contribution to the field.


Introduction
The primary focus of this paper is a new pan-genome method to determine core regions of a genome shared by all or almost all strains of the same species or subspecies. We evaluate the performance of this approach relative to other methods using experimentally determined essential genes under the hypothesis that all or at least most essential genes should be core across a species. This hypothesis implies that methods for determining core regions/genes are likely to be more accurate if they identify more essential genes as core genes. The paper reveals the potential usefulness of pangenome analysis for synthetic engineering and genome analysis more broadly through the analysis of core regions in Bacillus subtilis and Escherichia coli.
Over the last decade, considerable interest has been directed towards the determination of a minimal bacterial cell, making use of a short genome consisting of only essential genes for viability. The Mycoplasma mycoides JCVI-syn3.0 is a case example of synthetic engineering to design and build a genome that contains a streamlined gene set essential for cell viability and cell replication. 1 Multiple genome reduction projects have been undertaken. [2][3][4] More targeted genomic deletions of genomic loci have been performed to characterize essential genes, but generally targeted approaches are too laborious to perform on a whole genome. 5,6 However, the identification of "essential" genes -those genes that are critical for cell viability and replication -takes considerable time and effort in a laboratory setting and is usually determined with respect to one reference genome under one set of specific growth conditions. For instance, Kobayashi et al. 7 and Koo et al. 8 experimentally and computationally determined the minimal gene set in the Gram-positive bacterium Bacillus subtilis. Koo et al. 8 used a strictly experimental approach but Kobayashi et al. 7 used three forms of evidence for their essential genes as given in their Table 4: RB: previous experimental work in B. subtilis; RO: previous experimental work in other bacteria; and TW: their experimental work. The RO evidence used is a mix of experimental and computational as the determination of orthologs is computational and essentiality of those orthologs was not experimentally confirmed: "Through predictions we propose that 79 other genes are essential, whereas 106 are not (Table 3)". 7 Of the~4,100 genes of the type strain, a total of 271 genes for Kobayashi et al. 7 and 257 genes for Koo et al. 8 were shown to be essential. These essential genes were further categorized in terms of cell metabolism and enzymatic capability. Additionally, for the~4400 genes in the Gram-negative bacterium Escherichia coli, Goodall et al., 9 Baba et al., 10 and Yamazaki et al., 11 it was determined that 414 genes were essential to strain K-12.
Reuß et al. 3 completed extensive further experimental and computational work to determine a minimal B. subtilis genome they call MiniBacillus. They present a list of 523 protein coding and 119 RNA genes necessary for a minimal B. subtilis cell growing in complex medium at 37°C. While many of these genes are not essential under single deletion experimental conditions, they are required for survival because a cell needs certain essential functions which may be carried out independently by more than one gene. As noted by Reuß et al., 3 the choice of which functionally isologous genes to choose for a minimal cell depends upon minimization goals and gene choices for different functions are not independent of one another. One criterion used by Reuß et al. 3 is the conservation of the gene: "More strongly conserved genes were preferred over less conserved genes. In this respect, gene conservation and essentiality in genome-reduced Mycoplasma and other mollicutes species and the inclusion of genes in the genome of M. mycoides JCVI-syn3.0 had a high priority". Reuß et al. 3 do not explicitly use gene conservation at the species/subspecies pan-genome level but this seems in spirit with their criteria.
Reuß et al. 3 extended their computational prediction of MiniBacillus by building on previous work to generate B. subtilis strains with large genome reductions. 2 They started by constructing the delta 6 strain 12 (~8% genome reduction). This reduction removed: "two prophages (SPβ, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks)." The phage/prophage regions were identified in part by GC content and codon usage as a method to identify probable horizontally transferred regions. Pan-genome analysis was not used. Next, strain IIG-Bs20 was constructed from delta 6 13 by removing "all nine prophages, seven antibiotic biosynthesis gene clusters and two sigma factors for sporulation" in part to have a strain that would "not produce spores, antibiotics or bacteriocins". A direct descendant of IIG-Bs20, strain IIG-Bs27-47-24 (~31% genome reduction), was then used to generate more reductions in two REVISED Amendments from Version 1 Version 2 attempts to address all comments from the reviewers for version 1. This includes a new introductory paragraph explaining the overall thrust of the article before diving into a detailed background. A new paragraph better explaining the advantages of a pan-genome graph (PGG) over a simple pan-genome. A  Any further responses from the reviewers can be found at the end of the article independent strains, PG10 (~35% genome reduction) and PS38 (~36% genome reduction) 2 with the goal of removing genes "not necessary for the survival of the cell (e.g., sporulation, antibiotic production, motility, metabolism of secondary carbon sources, and genes of unknown functions)". For the strains IIG-Bs27-47-24, PG10, and PS-38, pangenome analysis was not explicitly used but one of several criteria for deleting genes was a lack of conservation across broader taxonomic groups.
For E. coli, Kolisnychenko et al. 5 generated an initial reduced genome in order to "serve both as a better model organism and as a more useful technological tool for genome science" by "deleting the largest K-islands of E. coli, identified by comparative genomics as recent horizontal acquisitions". K-islands are regions unique to the K-12 strain MG1655 compared with the O157:H7 strain Sakai, and the uroseptic E. coli strain CFT073. This comparative analysis with a limited set of genomes is an obvious precursor to pan-genome analysis with a much larger set of genomes. Umenhoffer et al. 14 generated the reduced E. coli strain MDS42 to be "free of mutation-generating IS elements". This approach does not rely on comparison to other strains, just the ability to identify IS elements. Csorgo et al. 15 further reduced the MDS42 strain by "constructing low-mutation-rate variants … to lack most genes irrelevant for laboratory/industrial applications." They targeted genes likely to be core and necessary for the species to adapt to the environment but detrimental in an industrial setting where strain stability is important.
Experimental studies to determine such essential genes are time consuming and often restricted to a single environmental condition using a single strain of the species. In addition, these approaches also knock out one gene at a time. As such, genes with multiple copies with redundant functions are often not considered as essential following knockout, as their additional copy is able to maintain cellular function. In other words, a viable organism would not result from deleting all but the experimentally determined essential genes from the genome. Another peculiarity of the single knockout essential genes is that pairs or cassettes of genes which can be removed and still have a viable organism are labeled essential because removal of just one gene is lethal. For example, removing the methylation gene(s) without removing the restriction digestion enzyme genes from the restriction mechanism results in cell death but the cell survives if the entire system is removed. This is likewise true for toxin/antitoxin systems.
While it is possible to define "essential" genes relative to viability, another larger question remains; which genes define a species? While specific phenotypes can vary across strains, in general a species seems to require some minimal set of genes to not only survive in the laboratory but to thrive in its natural environment. In contrast, some strains may have retained or acquired some genes which improve survival for specific niches. Comparing the genes from multiple diverse strains of a species can help answer these questions. We define the pan-genome for a species/subspecies to be the set of predicted orthologous gene clusters (OGC) across that set of strains. Others have allowed paralogs to be included in these gene clusters [16][17][18] but here we do not. This constraint forces there to be at most one gene per genome in an OGC.
We further define a pan-genome graph (PGG) to be a graph with the pan-genome OGCs as nodes where an edge exists between two nodes if the respective genes for any genome from the two OGCs are adjacent in that genome. More precisely, an OGC node is represented as a dipole with 5 0 and 3 0 ends and the edges go between an end of one node (5 0 or 3 0 ) to an end of another node depending on the orientation of the genes which are adjacent. The edges primarily represent the order and orientation of OGCs in the pan-genome genomes. Secondarily, the edges also represent the interstitial DNA sequences between the genes. A PGG edge has a weight equal to the number of genomes which contain the indicated adjacent gene ends. Core OGCs are defined to be those OGCs present in some large percentage of the strains in the pangenome (≥95% in this work). Core edges are defined similarly. Core regions are defined to be the coordinates in a genome for each set of adjacent core genes in that genome provided the edges between the core genes are also core edges.
The PGG is important as it captures the structure of the pan-genome in ways that simply treating the pan-genome as a set of OGCs cannot. The inherent gene context in the PGG allows for more accurate annotation of a novel genome than OGCs alone which struggle to differentiate recent paralogs/repeats. The PGG allows core regions to be defined for any genome rather than just core OGCs/genes. The PGG indicates which OGCs occur in cassettes with implications for function, evolution, and synthetic engineering. As discussed later, the PGG allows for determination of probable orthologs not captured in the OGCs.
Core OGCs should determine the baseline phenotype (capabilities and traits) of a species. Previous pan-genome studies 19 have shown that species tend to only tolerate the placement of noncore genes between core regions and not within those core regions. The reason an organism might constrain a core region rather than just core OGCs is that the region may include regulatory mechanisms such as operons, which allows for co-expression of multiple functionally associated genes, or regulons which would be disrupted with the insertion of other genes. We believe that conservation of core regions in species indicates resistance to insertion or deletion of genes in these regions through evolution or through human-mediated genetic engineering.
Here we present a pan-genome based calculation of core regions for B. subtilis ssp. subtilis and for E. coli. These core regions are compared with previous experimentally determined essential genes from the literature. These core regions are not a replacement for experimentally determined essential genes, but rather provide complementary information about a much larger portion of the genome. We expect that all truly essential genes for the species/subspecies would be a subset of the core OGCs/regions, since core OGCs would encompass genes responsible for providing a fitness advantage in environmental conditions as well as being essential for viability. This approach automates computational prediction of core OGCs/regions which can be used to help guide the removal of genome regions not needed for species fitness and indicate which genome regions are amenable to engineered insertions. This approach is an incremental improvement over previous computational methods to aid genome engineering. Ortholog prediction 17,18,20 and determination of genes essential for most bacteria has a long history. 21,22 Computational prediction of nonessential genes via predicting prophage regions or other horizontal transfer events is also well established. [23][24][25] Pan-genome tools, most of which at some level predict core genes, are also not new. 26,27 Our method builds directly upon our previous pan-genome work [28][29][30] and includes several improvements: 1) being able to use only complete high-quality genomes (this concept is not new, but we find it impacts the quality of the PGG and core region determination and is reasonable as more complete genomes become available); 2) checking for including the correct species/subspecies using average nucleotide identity (ANI); 3) reannotating gene features using homology and gene context to ensure consistency; and 4) generating a PGG for a rigorous definition of a core region. Figure 1 shows the high-level view of our method with details provided in the Methods section.

Genome Selection
Reference B. subtilis ssp. subtilis and E. coli genomes were selected for pan-genome construction using a series of filtering steps resulting in high-quality, non-redundant genome datasets (Table 1). For B. subtilis ssp. subtilis and E. coli,  we selected strains with complete genomes in RefSeq. 31 We restricted our analysis to complete genomes to ensure that missing genes due to incomplete genome sequencing/assembly did not affect the approach or results. We limited our choice to RefSeq for two reasons: RefSeq performs a series of quality checks to remove dubious genome assemblies, and the initial pan-genome construction depends upon reasonably consistent annotation which RefSeq provides. We extracted the genomes based on organism name: Bacillus subtilis (we did not specify subspecies, since for many RefSeq genomes a subspecies is not given) and Escherichia coli (we also specified Shigella since all Shigella species are actually considered to be the same species as Escherichia coli). 32,33 For each pan-genome, we then compared the genomes using a fast Average Nucleotide Identity (ANI) estimate generated using the MASH distance subtracted from 1 and multiplied by 100. 34 We used type strains and ANI to determine which of these genomes were the desired organism. We also used ANI to remove very closely related strains to reduce oversampling bias (for example for the B. subtilis type strain, 168, has at least eight genomes in RefSeq). We used GGRaSP 28 to choose a single medoid sequence from any complete linkage ANI cluster with a threshold of 0 The minimum pairwise ANI of any of the 1096 genomes was 95.53% which is not as tight as for B. subtilis ssp. subtilis which is to be expected given that E. coli is a species grouping not a subspecies grouping. One could arbitrarily try to choose a tighter grouping around the K-12 reference genome but the pairwise ANI values of the other genomes compared with the K-12 reference genome vary continuously from 96.22% to 100% with no punctate break in the values. After removing redundant genomes (using GGRaSP as discussed above), 969 E. coli genomes remained. We added back in two redundant genomes: The K-12 Entrez E. coli reference strain MG1655 (BioSample SAMN02604091) and the K-12 strain BW25113 (GenBank sequence accession CP009273.1, GenBank Assembly accession ASM75055v1/ GCA_000750555.1, GenBank BioSample accession SAMN03013572) used by Goodall et al. 9 These two redundant genomes were added back in so that we could map the PGG OGCs to these genomes for comparison to the established literature resulting in 971 genomes in the PGG (Table 1). By using a 95% threshold for the number of genomes an OGC must be in to be considered core, some small number of the 971 genomes could be engineered to remove what are normally core OGCs and not affect the assignment of core OGCs.

Pan-genome and PGG construction
For B. subtilis ssp. subtilis and E. coli, initial pan-genomes were based on the RefSeq annotation of these genomes. The pan-genome was generated using the pan-genome pipeline at the J. Craig Venter Institute (JCVI) at the nucleotide level using default parameters with the exception that a minimum of 90% identity and 90% length for pairwise BLAST matches were used to prevent possible clustering of non-orthologous genes. 29 This produced OGCs using gene context 30 as well as a PGG. 19 The PGG has two main components: nodes representing OGCs, and edges representing the sequence between OGCs and the order and orientation of the OGCs in the genomes. We updated the code repository for the JCVI pangenome pipeline with a script: iterate_pgg_graph.pl, which calls pgg_annotate.pl for the genomes in the existing PGG in order to ensure consistent annotation of the genomes and iterates until the PGG stabilizes. The script pgg_annotate.pl uses an existing PGG to assign regions of a genome to nodes of the graph. This is done by searching the medoid sequence using BLAST for the OGC the node represents against the genome and then uses Needleman -Wunsch 35 to extend the alignment if needed. If there are conflicting BLAST matches, then the matches are resolved based on which matches are consistent with the structure of the PGG which encapsulates gene context across the entire pan-genome. Once the nodes of the PGG are mapped to each of the genomes in the pan-genome a new version of the PGG is intrinsic and then explicitly extracted. This process is iterated to stability. This ensures that each genome is consistently annotated so that genes missing from the original annotation of some genomes will be consistently annotated across all genomes. A user manual for this new functionality is available at https://github.com/JCVenterInstitute/PanGenomePipeline.
Core regions were determined based on the PGG. Nodes in the PGG were OGCs. Edges in the PGG represented adjacency of genes (contained in the OGCs) in the underlying genomes. The definition of which OGCs were or were not considered "core" was determined relative to a threshold criterion. We used a criterion for core such that 95% or more of the underlying genome had to contain the OGC or edge. Considering that we used only complete genomes it might have been possible to use a 100% threshold. However, we opted for a 95% threshold based on prior experience and an abundance of caution to not under call core OGCs/edges which might result in false negatives. Each core region began with a core OGC followed by a core edge (if possible, otherwise the core region comprises a single OGC) to another core OGC and so on until a core edge cannot be found to continue the core region. A core region is just a path in the PGG which was then mapped onto each genome to determine the core region coordinates. When the core threshold was below 100% any genome may be missing an OGC (gene) or edge along this path which results in the path being broken into its remaining constituent parts.
Comparison to essential genes In order to compare core regions to experimentally determined essential genes we needed a common base of reference. For each of the experimental studies, the genes are specified based on a reference strain that was used for the experiments and has a complete genome in RefSeq. For Kobayashi et al., 7 only gene symbols/names were given which we mapped to Entrez GeneIDs using Entrez search. GeneIDs with no matches were manually curated to estimate the best matching gene symbol listed in the literature. For Koo et al., 8 locus IDs were provided giving direct access to the gene coordinates for RefSeq accession NC_000964.3 (BioSample SAMEA3138188, Assembly GCF_000009045.1). For Goodall, we used the data from three studies in Table S2 from Goodall et al. 9 Gene symbols/names again were all that was available but these were consistent with the GenBank annotation downloadable in gff format for the K-12 BW25113 reference genome (GenBank accession CP009273.1) used by Goodall et al. 9 (BioSample SAMN03013572). This gave us coordinates for all essential genes on RefSeq genomes which were annotated with a PGG which produces a file with coordinates for OGCs and edges mapped to the genome. These coordinates allow us to affiliate essential genes to OGCs.

Results
The original and refined PGG statistics for B. subtilis and E. coli are provided in Table 2. The major goal of refining the PGG using reannotation and iteration until stabilization was to achieve consistent annotation across all genomes in the PGG leading to a more comprehensive and cohesive PGG. While the RefSeq annotations of these genomes tends to be highly consistent, many small genes are often arbitrarily called from genome to genome and even some common longer genes can occasionally be missed. There are three obvious points of improvement in the refined PGG for both the OGC and edge stats: the number of size 1 OGCs/edges significantly decreased due to some dubious RefSeq gene calls being eliminated and some becoming shared with other genomes; the number of core OGCs/edges significantly increased showing an improvement in the consistency of annotation across all genomes; and the number of genes/edge instances in OGCs/edges greatly increased again indicating a much more consistent annotation. We have included core OGC statistics for three threshold definitions of core: 95%, 99%, and 100%. In part, this is for comparison to previous studies but it also illustrates the relative larger impact of consistency as the threshold increases. For example, in E. coli, the refined PGG gives an increase of 22% in core OGCs at a 95% threshold but an increase of 111% in core OGCs at a 100% threshold. When even a single misannotated gene drops an OGC below core at the 100% threshold consistent annotation is crucial.  Table 3). The 289 essential genes which are core OGCs are contained in only 63 of these regions. These 289 essential genes are not evenly distributed in these 63 regions (e.g. 46 are in core region 3). Similarly, the 16 essential genes in non-core regions (the regions between core regions) are contained in only seven noncore regions with eight genes in the non-core region between core regions 206 and 207 ( Figure 2 Table 1). They include all 30 rRNA and 86 tRNA genes from the reference genome as well as three "misc" RNA genes in MiniBacillus. The three misc RNA genes are present in all 108 genomes. In all likelihood, the 10 copies of the 16S-23S-5S RNA operon are not required but it is safer for robust growth not to delete any of them. Likewise, for the tRNA genes where many are redundant. For the 30 rRNA genes: six are noncore (92-102 genomes), 16 are core but not in all genomes (103-107), and eight are in all 108 genomes. It is clearly possible that some of these strains are dispensing with some of the RNA operons but at most this is happening rarely reinforcing the decision not to remove any from MiniBacillus. In addition, some of the missing RNA operon genes may be due to incorrect assembly of the two sets of tandem RNA operons (one a two-unit tandem and one a three-unit tandem) as large tandem repeats can be problematic for assemblers. All the rRNA genes in fewer than 106 genomes are in the tandem rRNA operons (Supplementary Table 4). Of course, the tandem rRNA operons are the most likely to be deleted via recombination as well. For the 86 tRNA genes: 13 are noncore (100-102 genomes), 18 are core but not in all genomes (103-107), and 55 are in all 108 genomes. Retaining all the tRNA genes in MiniBacillus also seems to be the correct decision as strains rarely dispose of the tRNA genes.
Both the experimentally determined essential genes and the predicted core OGCs/regions are important data for genome engineering.
They both indicate regions that should not be deleted without careful consideration. The noncore regions also indicate where the bacterium is more likely to tolerate engineered insertions. As a validation of our method and how to interpret the results our method produces it is important to understand why 16 essential genes are in noncore regions.
For B. subtilis, both Kobayashi et al. 7 and Koo et al. 8 used similar single knockout methods to determine "essential" protein-coding genes when grown in LB at 37°C. Koo et al. 8 identified 257 essential genes while Kobayashi et al. 7 identified 271 essential genes. The union of these two sets results in 305 essential genes (Supplementary Table 2 37 Gene rttF/yqcF (OGC 4590 present in 46 of 108 genomes) and gene rtbE/ yxxD (OGC 4772 present in 53 of 108 genomes) are also the antitoxin of a cognate toxin-antitoxin pair. 38 Gene yezG (OGC 4411 present in 43 of 108 genomes) is also the toxin for a cognate toxin-antitoxin pair. 39 Gene sknR/yqaE (OGC 4643 present in 34 of 108 genomes) is part of a phage-like region which, if removed would still allow B. subtilis to remain viable 12 possibly because it is another antitoxin or similar mechanism. Genes bsuMA/ydiO (OGC 4838 present in 24 of 108 genomes) and bsuMB/ydiP (OGC 4839 present in 24 of 108 genomes) are part of a prophage region of about 15 genes in 48 genomes which includes ydiR and ydiS which are type-2 restriction enzymes. These are not essential genes, but they are essential if the restriction enzymes are present. 40 We are not the first to notice these issues with experimentally determined essential genes indicated by our references above. In their review, Commichau et al. 36 referred to these as "protective essential genes." In fact, Koo et al. 8 also addressed this in their paper: "Of the 257 genes essential in LB medium, 30 are not essential in some other growth condition or genomic context...LB may have an insufficient amount of particular compounds; e.g., the ylaN mutant requires a higher amount of iron than that present in LB … or may lack a compound that could bypass the need for that gene product; e.g., eno, pgm, gapA, and alrA … Some gene products are essential only at high growth rates typical of LB at 37°C (smc and scpA …), and these may not be essential in the natural soil environment where B. subtilis grows slower. Finally, some genes are non-essential in specific genetic backgrounds, e.g., antitoxins can be deleted in strains lacking their cognate toxin gene". (OGC 4748 present in 35 of 108 genomes), tagA (OGC 4749 present in 35 of 108 genomes) and tagB (OGC 4750 present in 35 of 108 genomes). The WTA genes are involved in production of anionic glycopolymers required for consistent cell shape and division. 41 The WTA genes are part of a 31 gene region which has been shown to be dispensable 42 but results in malformed cells with poor growth properties. Gene rodA (OGC 3994 present in 97 of 108 genomes) appears to be the exception as it is asserted to be essential for maintaining a rod shape and preventing spherical cells which lyse. 43 Kobayashi et al. 7 stated: "Ten essential genes are involved in cell shape and division. Septum formation requires seven (ftsA, L, W, and Z, divIB and C, and pbpB …), whereas cell shape requires three (rodA, and mreB and C)." Interestingly, genes ftsZ (OGC 1675 present in 105 of 108 genomes) and pbpB (OGC 1662 present in 107 of 108 genomes) while considered core, using our 95% of genomes definition are the only core OGCs not present in all 108 genomes. We investigated these 11 genes further to understand why essential genes did not appear to be core OGCs. By examining the PGG we discovered that alternate OGCs with homology to the essential genes had replaced the essential genes. Gene pbpB (OGC 1662 in 107 genomes) is replaced in the one remaining genome by OGC 7120 which is also annotated as As an illustrative example for rodA, Figure 3 shows how this is represented in the PGG. The medoid sequences for OGCs 6436 (A4A60_RS20560), and 8718 (C7M30_RS12210) have full length homology to the medoid sequence for rodA (OGC 3994, ETA10_RS20040) with 66% nucleotide /65% peptide and 83% nucleotide/85% peptide identity respectively. For B. subtilis ssp. spizizenii strain W23, poly (ribitol phosphate) is the main teichoic acid 44 and this was thought to distinguish ssp. spizizenii from ssp. subtilis whose type strain 168 has poly (glycerol phosphate) as the main teichoic acid. Further study found that the ribitol/glycerol distinction does not distinguish between spizizenii and subtilis subspecies 45 but rather either subspecies can contain one or the other. Our PGG confirms this and in fact finds six distinct variants of the WTA region. For example the tagD gene (OGC 4748 in 35 genomes) has been replaced by multiple orthologs with the same annotation: OGC 3746 (23 genomes), OGC 5431 (43 genomes), OGC 6915 (two genomes), OGC 7624 (three genomes), and OGC 8731 (one genome). The variation of the WTA region in B. subtilis will be the focus of a future paper. 46 For the 34 protein coding genes from MiniBacillus 3 which were not in all 108 genomes (Supplementary Table 6), 10 were already discussed above as to why they were essential but not core. The seven essential genes previously shown to be protective essential genes are as expected not in the MiniBacillus data set. The noncore tuaB gene was essential in both data sets but not included in MiniBacillus. This leaves 24 MiniBacillus protein coding genes which are noncore and unexplained. The tagU and gtaB genes are part of the WTA cassette discussed above. The four fecC-F (also called yfmC-F) genes form a cassette and are in 98 genomes. From Reuß et al.: 3 "For iron uptake, the minimal cell should possess the EfeUO system for elemental iron uptake and the iron-citrate ABC transporter YhfQ-YfmCDEF (136, 137)." (yhfQ is present in all 108 genomes) but no alternate mechanism is specified. (2) (97)  Table S1. Strains PG10 and PS38 were derived from strain IIG-Bs27-47-24 which in turn was derived from strain delta 6. This means all deletions in delta 6 are present in the other strains, and all deletions in IIG-Bs27-47-24 are present in PG10 and PS38. For delta 6, most of the deleted genes are noncore which would be expected since most of the deleted regions were phage/prophage regions (Table 3). For additional deletions to IIG-Bs27-47-24, almost a quarter of the deleted genes are noncore which would again be expected as more prophage and horizontally transferred regions were intentionally targeted but now more core genes were deleted based on core functionality deemed not to be essential for laboratory growth such as sporulation (Table 3). For additional deletions to PG10 and PS38, most deleted genes were core as most of the obviously horizontally transferred regions had already been deleted (Table 3). While pan-genome analysis was not used to select the deleted regions, we believe it could have provided strong evidence to support the deletion of the noncore genes/regions which were deleted. In addition, it could be used to suggest further deletions. There are nine noncore regions which contain seven or more noncore genes which have not yet been deleted in any of these strains ( Table 4). The largest of these regions contains the WTA genes cassette we discussed above and is not a good candidate for deletion. By examining the refined PGG at these regions it is straightforward to determine if there are alternate OGC choices for the region that in sum designate the region as likely to be core as we showed in Figure 3.
To show that our method produces significantly different results than previous methods we compared our pan-genome analysis to the very recent work on a B. subtilis pan-genome by Wu et al. 47 While the focus of Wu et al. 47 was on determining which genomes should be excluded from a species/subspecies pan-genome based on "incorrectly classified Bacillus subspecies strains, phylogenetically distinct strains, engineered genome-reduced strains, chimeric strains, strains with a large number of unique genes or a large proportion of pseudogenes, and multiple clonal strains", their analysis focused on how this affected the determination of core OGCs. We compared our core OGC set to theirs for the reference genome. Wu et al. 47 discussed two pan-genome data sets: "old (89 strains) and new (153 strains)". We compared to the new data set which is more recent and more comparable to our pan-genome of 108 strains (Supplementary Table 5). After removing "confounding" strains the new data set had 128 strains. From their Table 1 compared to our Table 2, Wu et al. 47 have many fewer core OGCs whether defined at 95%, 99%, or 100% both for our original and refined PGGs. We compared their methods to ours to attempt to account for the difference. They also apparently restricted genomes to those available from RefSeq since they mention a RefSeq ID. They did not require the genomes to be considered complete by RefSeq as we did but instead used these criteria: "Among these B. subtilis strains, we removed strains whose N base content was greater than 1% of the genomic size (FB6-3,GS 188, SR1), and we removed the chimeric genome BEST7613 with a genome size of 7.6 Mb.". We used the RefSeq annotation which is generated by a consistent NCBI annotation pipeline. They also tried to ensure consistent annotation: to "ensure the consistency and reliability of the annotation and gene prediction of the genome, we used the program Prokaryotic Genome Annotation System (Prokka)". We doubt the different annotations from these two established pipelines accounts for many differences in core OGCs. Both methods used a whole genome ANI method to discard outlier genomes. There are multiple differences in our pan-genome approach. First, we used PanOCT and they used Roary. Second, we used all annotated gene features: gene (protein coding), pseudogene, miscRNA, rRNA, and tRNA, whereas they used only protein-coding genes. Finally, and we think most importantly, we iterated over annotating the genomes and PGG refinement to ensure consistent annotation and they did not. To see what impact our choice of all gene features versus just protein-coding genes had we looked at the annotation of core OGCs on the reference genome (Supplementary Table 1). Luckily all 3778 core (95% threshold) OGCs are present in the reference genome. Of these, 3473, 3334, and 3189 are protein coding OGCs at thresholds 95%, 99%, and 100% respectively. All these numbers are still much higher than those reported by Wu et al. 47 We should note that even though we did not count the 25 core OGCs annotated as pseudogenes in the reference genome, some of the core protein-coding OGCs in the reference genome might be annotated as pseudogenes in other genomes which could impact the Wu et al. 47 numbers. Roary tends to require near full length gene matches which is why we required PanOCT to only use 90% or longer length matches. The authors chose to limit Roary to 95% identity or higher matches which we think is much too high since the species ANI threshold is 95% and even subspecies ANI threshold of 98% is too close to this threshold given that some genes are more rapidly evolving than others so we used a threshold of 90% or higher identity for matches. Even with our 90% identity threshold some genes such as rodA, discussed above, drop below this threshold generating possibly unnecessary branching in the PGG. Of the 128 strain pan-genome from Wu et al. 47 that we compared to our 108 strain pan-genome, 92 strains were in common with 16 being exclusive to our pan-genome and 36 being exclusive to theirs. Of the 36 strains exclusive to theirs 23 were removed as being redundant at the ANI level by us, 9 were in RefSeq but not complete genomes, and 4 either were never in RefSeq (they do not have RefSeq IDs in their Supplementary Table 3) or no longer are. Interestingly, while 15 of the 16 genomes exclusive to ours are just more recent strains to RefSeq, one strain, D12-5, was used by us but discarded by them. They discarded D12-5 because "BS155 and D12-5 possess the largest proportion of pseudogenes (37.96% and 11.32%) among the B. subtilis strains" and for D12-5 they indicated this was due to a large number of frameshifts. Pseudogenes due to frameshifts are often an indication of lower quality assembly consensus sequence from using only long reads at lower coverage. Our pan-genome method is resilient to this kind of error profile in the genome due to reannotation of the genomes and PGG refinement whereas other pan-genome methods are not. We believe our higher counts for core protein coding OGCs is correct. To validate this, we compared how many of the 305 essential B. subtilis genes are core for both methods. For the 18 genes we discussed above that are essential but not in all 108 genomes of our PGG, 2 are core at 95% and 1 is core at 99%; whereas, for Wu et al. 47 2 are core at 95%, 2 are core at 99%, and 1 is core at 100%. The only significant difference for these 18 genes is that ftsZ is in 95% (105) of our pan-genome and 100% of theirs. The Wu et al. 47 pan-genome misses many additional essential genes which ours does not: 28, 39, and 47 for 95%, 99%, and 100% thresholds respectively.
For E. coli, Goodall et al. 9 determined E. coli essential genes using an analysis of transposon insertion events (TraDIS). The results of their study and two other studies, the Keio collection 10 and the Profiling of the E. coli Chromosome (PEC) 11 were captured in Table S2 of Goodall et al. 9 Of the 414 genes with overlap between these studies, the 248 essential genes in common for all three studies are all core OGCs (Figure 4 and Supplementary Table 7). This set of 248 essential genes should be the highest quality predictions as determined by all three studies and confirms our assertion that essential genes should almost always be core OGCs. The next highest quality set of essential gene predictions is the 45 essential genes where two of the three studies agree which 41 are core OGCs: for Keio-PEC, 15 of 16 are core OGCs; for TraDIS-Keio, eight of 11 are core OGCs; and for TraDIS-PEC, 18 of 18 are core OGCs  (Supplementary Table 7). The lowest quality set of essential gene predictions is the 121 essential genes where only one  study agrees which 89 are core OGCs: for Keio only, 12 of 22 are core OGCs; for PEC only, 18 of 18 are core OGCs; and  for TraDIS only, 59 of 81 are core OGCs (Supplementary Table 7). One of the noncore essential genes present in two studies (TraDIS-Keio), racR, is probably a toxin suppressor which is not essential in the absence of the toxins. Bindal et al. 48 noted, "We further show that both YdaS and YdaT can act independently as toxins and that RacR serves to counteract the toxicity by tightly downregulating the expression of these toxins". The racR gene is found in only 106 of the 971 genomes in the E. coli PGG, whereas ydaS and ydaT are found in 106 and 150 genomes respectively, perhaps arguing that ydaS is the key toxin gene. This recapitulates the pattern we observed in B. subtilis where toxin suppressor genes are only essential in the presence of toxin genes. Similarly, the dicA gene (TraDIS-Keio) can be deleted if the dicB gene is also deleted. Kato et al. 49 noted: "The dicA gene encoding a repressor of a cell division inhibitor was deleted in our study with the dicB, the inhibitor gene". There are 521 core regions for E. coli (Supplementary Table 8). The 378 essential genes which are core OGCs are contained in only 133 of these regions. These 378 essential genes are not evenly distributed in these 133 regions (e.g., 27 are in core region 362). Similarly, the 36 essential genes in non-core regions (the regions between core regions) are contained in only 23 non-core regions with four in the non-core region between core regions 152 and 153. A table of all E. coli genes mapped to the reference is provided in Supplementary  Table 9.
Yang et al. 50 presented a similar pan-genome analysis for 491 E. coli strains. There were 420 strains in common between the Yang et al. 50 491 strain pan-genome and our 971 strain pan-genome (Supplementary Table 10). Our pan-genome included Shigella species (see Methods) which Yang et al. 50 did not. This added diversity of our pan-genome should reduce the number of core OGCs. Likewise, the much larger number of strains in our pan-genome should reduce the number of core OGCs. Yang et al. 50 report 867 core protein-coding genes presumably at a 100% threshold although this is not explicitly stated. For our refined PGG, we had 1501 core OGCs at the 100% threshold. We include all genes in our OGCs but 1234 of the 1501 core OGCs are protein coding at the 100% threshold. Yang et al. 50 did not provide a table of their core genes for sake of comparison, however we expect for the same reasons as for our more detailed analysis of the B. subtilis pan-genome that our set of core OGCs is more complete. Yang et al. 50 reported that their core genes included 243 essential genes from the DEG database 51 which contains essential genes from many studies but did not provide a There is of course no "gold standard" that provides a 100% correct set of core regions/genes for a pan-genome/species. When comparing our method to others, this leaves only indirect measures of accuracy. We compared our method versus two other recent core gene determinations for Bacillus subtilis and Escherichia coli and showed that our method was superior using coverage of essential genes by core genes as an indirect measure. We also showed that the PGG allowed for a detailed analysis of exceptions such as when an OGC is replaced by a more distant ortholog.

Discussion
For the purpose of biological engineering, determining the set of core regions for a given species is critical as changes to these regions should be expected to reduce fitness or be lethal. Core regions indicate parts of the genome that are conserved across evolution within a species. These regions are not necessarily required for survival but presumably confer a fitness advantage and define the characteristic core genotype which produces the core phenotype (lifestyle).
Since most essential gene studies are carried out under specific static laboratory growth conditions, genes which would normally be essential for a species across a diverse set of dynamic environmental conditions might not be discovered (e.g., necessary for fluctuating temperatures). Correspondingly, genes required to out compete rival organisms through increased fitness or to evade immune responses might not be found under laboratory conditions are considered facultative essential. 31 Core regions, therefore, should be a superset of essential genes in most cases but exceptions might occur for genes which are not needed in a species' natural niche but are required in a laboratory setting. Another exception would be for genes which are essential for a particular strain but not for other strains due to the presence of compensating non-core genes.
Noncore OGCs/regions which are determined by pan-genome analysis are often horizontally transferred elements, such as phage, prophage, or mobile elements. For industrial applications these regions are dispensable and can even be sources of genome instability. 3,[12][13][14]53 While there are other methods for identifying these regions, pan-genome analysis is a reliable complimentary tool. Pan-genome analysis can also reveal enzymatic and other systems/pathways that are present in some strains but not others 53 which indicates they can likely be removed. When choosing between retaining alternate systems for essential functions, biological engineers have looked at conservation of those systems across broad taxonomic levels 3 as an indication of utility and we believe conservation across the pan-genome should also be considered. When specific genes/systems of known function are being targeted for removal pan-genome analysis is less useful but still good information to have. For instance, Reuß et al. 2 tried to delete region BSU07710-07820 from B. subtilis which was lethal. In this region, six of the 11 OGCs are core but the five noncore genes are adjacent so perhaps region BSU07750-07790 could have been successfully deleted.
Given that we believe pan-genome analysis is a useful complimentary tool for biological engineers, it is important that the pan-genome analysis used be as accurate and helpful as possible. We showed by comparing with other recent pangenome studies for B. subtilis and E. coli that our method is more accurate for determining core OGCs/regions as validated by coverage of essential genes. Further, we believe that the PGG is valuable for confirming when noncore OGCs may be compensated for with alternate homologous OGCs at the same relative genomic location performing the same function as we showed in Figure 3. The function of these noncore OGCs may be essential and should be considered appropriately.
Pan-genome studies often capture the diversity of sequenced species but fail to compare gene lists to experimentally validated essential genes lists or the results are confusing. Interestingly in Mycoplasma, fewer essential genes were determined with the pan-genome method compared with the laboratory experimental approach. 54 In Pseudomonas, only one-third of the pan-genome single copy genes had overlap with the essential genes from experimentally reduced genomic studies. 55 We showed that the core OGCs/regions from our refined PGG encompass 91% and 95% of the E. coli and B. subtilis experimentally determined essential gene lists, respectively. Both model bacterial species E. coli and B. subtilis have had many genome reduction studies performed and reviewed elsewhere. 56 Experimental verification of the essentiality of computationally predicted core OGCs or regions requires that each strain of the pan-genome study be minimized. However, it is cost prohibitive to do knockout studies on all strains of a pangenome. One must carefully choose a single genome as a representative of the entire pan-genome for the purpose of verifying the essentiality of core regions and/or the non-essentiality of noncore regions by experimental validation. However, given the diversity of most bacterial species it is unlikely that any one strain completely captures the capabilities of the species in all environmental conditions. Further, while there are clearly core OGCs/regions associated with viability for a species, other core regions probably contribute to a lesser degree to cell viability. For example, for the purpose of biological engineering, changes in these locations may reduce fitness by slowing cell growth.
The use of a PGG for identifying core regions of a bacterium is an automatable, low-cost, rapid, and effective way to evaluate both Gram-negative and Gram-positive bacteria. This method compliments and expands upon the experimental knockout approach by including environmental diversity as a measure of what regions and OGCs are conserved across the species. The approach also overcomes the limitations of knockout studies that are specific to the strains and growth conditions used.
The B. subtilis WTA region provides a cautionary note for relying entirely upon core regions to determine what is safe to remove. While most non-core regions involve cassettes of genes which are entirely absent from some strains such as phage regions, sometimes orthologous replacement possibly due to homologous recombination can have functionally equivalent genes appearing to be non-core. A closer examination of the PGG can determine if a region is simply missing from some strains versus being replaced in which case further study may be needed before removal of the region. Of course, in some cases the orthologous replacement does not need to occur at the same location in the genome but that was the case for all instances we examined in B. subtilis.
While we showed that almost all essential genes are core OGCs and most are OGCs at the 100% threshold, the exceptions are interesting. We discussed issues such as "protective essential genes" 36 (such as toxin/anti-toxin gene pairs) and more distant orthologs not captured in OGCs. We did not discuss genes which might be undergoing gene loss. 57 The PGG is well suited to looking at which subset of genomes have suffered a gene loss and possible mechanisms such as gene replacement. The PGG has been used to show which genomic regions tend not to allow insertions of horizontally transferred genes 19 and where metabolic cassettes can be swapped. 53

Acknowledgements
The authors would like to thank IARPA for sponsoring this research and would like to thank Derren Barken for his assistance in table generation. This project contains the following underlying data:

Data availability
• Table 1. Selection of complete genomes for B. subtilis and E. coli PGGs.
• Table 2. Pan-genome graph statistics for B. subtilis and E. coli.
• Table 3. The number of deleted genes from B. subtilis reduced strains which are noncore versus core.

Open Peer Review
between core and essential genes is indeed their primary hypothesis ("We expect that all truly essential genes for the species/subspecies would be a subset of the core OGCs/regions"). This should be more explicitly stated, perhaps in the first paragraph of the Introduction.
What advantage is provided by keeping the directionality of OGCs in the PGG? Is this purely a methodological checkpoint, i.e. improve the detection capability by reducing the number of false positive or negative hits, or is it further used in the analysis and interpretation of the results? Needs to be clarified, as it increases the complexity of the pan-genome turning a set into a graph. There is a passage "PGG refinement to ensure consistent annotation", which alludes to the actual role of PGG.

3.
Another general comment connected to the above, esp. major comment 2: the report serves a dual role as a software announcement (update) of JCVI's pan-genome pipeline software suite, with additional elements and certain conceptual advances, as well as the comparison of the core-vs-essential sets for two of the best studied/sampled species pangenomes. This should be a bit more clearly explained perhaps. The correspondence of core to essential genes is a welcome contribution but may not be the main topic of the manuscript, just a conclusion drawn from the analysis.

4.
Following major comment 4: the method does well in identifying core regions and indeed makes a convincing case for an improvement over other methods. Yet, the comparison with essential genes is an addition, but not a comparison against other methods that define core regions. As the authors decided to take this direction, as they improve over their own previous methodology, this point should be qualified appropriately. In other words, the 'improvement' can be shown as an incremental step over a previous protocol and explicitly shown that it is validated against 'essential' gene sets. If this point is not emphasized, the analysis will be seen as lacking a comparison to another 'gold-standard' method (experts know that there is no such thing, yet). Pages 11-12 have some elements of a comparison to another approach, this could be extended by a couple of concluding sentences. A good spot where some concluding remarks can be made might be a short paragraph before the Discussion.

Minor comments:
In Introduction: "We further define a pan-genome graph (PGG) to be a graph", this should probably follow the paragraph starting "Here we present a pan-genome based calculation..." ?
"to not under call core OGCs/edges", i.e. to reduce the number of potentialy false negatives. Or, increase coverage. 6.
for B. subtilis: "3419 (73.5%) core and present in all 108 genomes": this row in Table 1 should be somehow highlighted, perhaps by color or other means --it is an important part of the study and a key result. 7.
a word for missing genes in the context of potential gene loss and the possibility of including them in future steps (see PMID: 12840037 1 ); this is something we (and possibly others) have been trying to implement for pangenome data, without much success. Something to discuss as a partial explanation for 'key' (essential?) missing genes in certain lineages within the species pedigree, perhaps? 8.
"For the 34 protein coding genes"... good yet incredibly dense paragraph, a (supplementary) table might help here.

9.
Would the PGG implementation also help future studies in synteny analysis/conservation? Maybe a minor point that can be included in the discussion, with appropriate (1-2) references. A concluding short paragraph following the current one with the WTA region might be a good way to wrap up.

10.
Reviewer Expertise: Computational Biology, Biological Computation, Systems Biomedicine, Bioinformatics, Protein Structure I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
method is that it's now taking steps to ensure that 'bad genomes' are filtered out, and only the 'high-quality' ones are left.... Figure 1, 2nd line: "Compute genome ANI using Mash". This doesn't make sense, as ANI and Mash are different approaches. Mash does not estimate ANI (as it is a distance). Unless the authors took the distance and subtracted it from 1 before multiplying by 100, they do not have an approximate ANI (see fastANI paper [PMID: 30504855] 1 or the Mash paper [their ref . 34] where Mash and ANI methods are compared). Further, the authors state they use type strains and ANI (presumably using the Mash derived approximation which is not ANI) to remove very closely related strains but do not specify what criteria/value was used to determine very closely related strains. Since the authors chose to use a program (GGRaSP) that uses ANI matrixes as the input, it can be assumed that either ANI values were calculated by an unspecified method, or they used transformed Mash values to approximate ANI and need to specify this transformation earlier in the methods. Either way, this should be clearly stated in the methods section, and not leave the reviewer to guess how this might have been done.

Methods:
"The 132 genomes were reduced to 109 after removing…" -it is unclear what the condition for removal was. It would be helpful if this was explicitly stated (presumably an approximate ANI value between 95.73% and 97.28%). Also the authors state the minimum ANI between B. subtilis was 97.28% and the maximum ANI of any of the 11 other genomes to the 132 was 95.73%. The 11 genomes referenced here are unclear and the maximum ANI for the 132 is not provided. It is important to clearly bound their values, in order to enable comparison to other studies. For E. coli the parameters used to remove redundancy need to be explicitly stated and how the groups are collapsed (i.e. genomes A to genome B has 99% ANI value, which genomes is removed and which genome is retained?). The authors should explicitly state why they added 2 redundant genomes to the E. coli dataset but did not do similar additions for B. subtilis. While the PGG approach seems fairly good, the heavy reliance on RefSeq annotations could be problematic for other species.

Results:
The results shown in Table 1, and the bottom line is that for both B. subtilis and E. coli, the refined cores are a bit larger (and contain a larger fraction of 'essential genes' for the species). The E. coli core is about a third larger, going from 2200 to 3100. The latter number (3100) seems to be more consistent with what's expected for E. coli, based on many different experiments -historically, there has always been roughly 3000 E. coli genes. So from this perspective, 2218 genes seems a bit too small (and also some of the 'essential genes' were missing from the core.) I'm curious as to whether a non-RefSeq gene annotation tool (for example, Prokka) be utilized to improve the consistency of gene calls? The specific results with number breakdowns are very confusing to read on a first pass and require very careful reading to understand the somewhat odd notation being used. This should be cleaned up to enhance readability.

Discussion:
The discussion surrounding the issue of lab conditions and core regions is a good. In addition, the discussion around noncore OGCs/regions also shows how the proposed pan-genome analysis could be used to identify noncore regions that could be removed that experimental results have been unable to identify. It might have been good to have a brief discussion of the phylogroupspecific cores in E. coli [see PMID: 33500552 2 -disclaimer -this is a recent publication from our group.] The discussion section overall provides a good wrap up to the paper and summarizes how the PGG approach can be leveraged and the benefits from utilizing this approach.