SUMMARYSegmental duplications contribute to human evolution, adaptation and genomic instability but are often poorly characterized. We investigate the evolution, genetic variation and coding potential of human-specific segmental duplications (HSDs). We identify 218 HSDs based on analysis of 322 deeply sequenced archaic and contemporary hominid genomes. We sequence 550 human and nonhuman primate genomic clones to reconstruct the evolution of the largest, most complex regions with protein-coding potential (n=80 genes/33 gene families). We show that HSDs are non-randomly organized, associate preferentially with ancestral ape duplications termed “core duplicons”, and evolved primarily in an interspersed inverted orientation. In addition to Homo sapiens-specific gene expansions (e.g., TCAF1/2), we highlight ten gene families (e.g., ARHGAP11B and SRGAP2C) where copy number never returns to the ancestral state, there is evidence of mRNA splicing, and no common gene-disruptive mutations are observed in the general population. Such duplicates are candidates for the evolution of human-specific adaptive traits.

This work comprehensively characterizes lipidomic changes underlying daptomycin resistance in three Gram-positive bacterial species, E. faecalis, S. aureus, and C. striatum, by using a novel three-dimensional lipidomics methodology based on advanced mass spectrometry. We demonstrated a number of advantages of our method in comparison with other methods commonly used in the field, such as high molecular specificity, sensitivity, and throughput. Whole-genome sequencing of the S. aureus and C. striatum strains identified mutations in pgsA, which encodes phosphatidylglycerophosphate synthase, in both resistant strains. Lipidomics revealed significantly decreased levels of lipids downstream of PgsA, as well as accumulation of lipids upstream of PgsA in the resistant strains. Furthermore, we found that changes in individual molecular species of each lipid class depend on the their specific fatty acid compositions. The characteristic changes in individual lipid species could be used as biomarkers for identifying underlying resistance mechanisms and for evaluating potential therapies.

While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI. While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.

Genetic differences that specify unique aspects of human evolution have typically been identified by comparative analyses between the genomes of humans and closely related primates1, including more recently the genomes of archaic hominins2,3. Not all regions of the genome, however, are equally amenable to such study. Recurrent copy number variation (CNV) at chromosome 16p11.2 accounts for ~1% of autism cases4,5 and is mediated by a complex set of segmental duplications, many of which arose recently during human evolution. We reconstructed the evolutionary history of the locus and identified BOLA2 (bolA family member 2) as a gene duplicated exclusively in Homo sapiens. We estimate that a 95 kbp segment containing BOLA2 duplicated across the critical region ~282 thousand years ago (kya), one of the latest among a series of genomic changes that dramatically restructured the locus during hominid evolution. All humans examined carry one or more copies of the duplication, which nearly fixed early in the human lineage—a pattern unlikely to have arisen so rapidly in the absence of selection (p < 0.0097). We show that the duplication of BOLA2 led to a novel, human-specific in-frame fusion transcript and that BOLA2 copy number correlates with both RNA expression (r = 0.36) and protein level (r = 0.65), with the greatest expression difference between human and chimpanzee in experimentally derived stem cells. Analyses of 152 patients carrying a chromosome 16p11.2 rearrangement showed that >96% of breakpoints occur within the Homo sapiens-specific duplication. In summary, the duplicative transposition of BOLA2 at the root of the Homo sapiens lineage ~282 kya simultaneously increased copy number of a gene associated with iron homeostasis and predisposed our species to recurrent rearrangements associated with disease.

Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132, derived from Enterococcus faecalis, as being capable of integrating single-stranded DNA oligonucleotides into the S. aureus genome. We found that EF2132 can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some S. aureus strains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the S. aureus genome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in S. aureus which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds.

BACKGROUND: Microsatellite instability (MSI) is an emerging, actionable phenotype in oncology that informs tumor response to immune checkpoint pathway immunotherapy. However, there remains a need for MSI diagnostics that are low cost, highly accurate, and generalizable across cancer types. We developed a method for targeted high throughput sequencing of numerous microsatellite loci with pan-cancer informativity for MSI using single-molecule molecular inversion probes (smMIPs). METHODS: We designed a smMIP panel targeting 111 loci highly informative for MSI across cancers. We developed an analytical framework taking advantage of smMIP-mediated error correction to specifically and sensitively detect instability events without the need for typing matched normal material. RESULTS: Using synthetic DNA mixtures, smMIPs were sensitive to at least 1% MSI positive cells and were highly consistent across replicates. The fraction of identified unstable microsatellites discriminated tumors exhibiting MSI from those lacking MSI with high accuracy across colorectal (100% diagnostic sensitivity and specificity), prostate (100% diagnostic sensitivity and specificity), and endometrial cancers (95.8% diagnostic sensitivity and 100% specificity). MSI-PCR, the current standard-of-care molecular diagnostic for MSI, proved equally robust for colorectal tumors, but evidenced multiple false negative results in prostate (81.8% diagnostic sensitivity and 100% specificity) and endometrial (75.0% diagnostic sensitivity and 100% specificity) tumors. CONCLUSIONS: smMIP capture provides an accurate, diagnostically sensitive, and economical means to diagnose MSI across cancer types without reliance on patient-matched normal material. The assay is readily scalable to large numbers of clinical samples, enables automated and quantitative analysis of microsatellite instability, and is readily standardized across clinical laboratories.

Structural variation and single-nucleotide variation of the complement factor H () gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ∼360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four -related () gene paralogs ( and ∼25-35 Mya and and ∼7-13 Mya). Remarkably, all evolutionary breakpoints share a common ∼4.8-kbp segment corresponding to an ancestral gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral , creating four fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of [ = 5.81 × 10, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD ( < 10) and AHUS ( = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new genes but also in the predisposition to complex human genetic disease phenotypes.

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