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The genomes of living lungfishes can inform on the molecular-developmental basis of the Devonian sarcopterygian fish-tetrapod transition. We de novo sequenced the genomes of the African (Protopterus annectens) and South American lungfishes (Lepidosiren paradoxa). The Lepidosiren genome (about 91 Gb, roughly 30 times the human genome) is the largest animal genome sequenced so far and more than twice the size of the Australian (Neoceratodus forsteri)1 and African2 lungfishes owing to enlarged intergenic regions and introns with high repeat content (about 90%). All lungfish genomes continue to expand as some transposable elements (TEs) are still active today. In particular, Lepidosiren's genome grew extremely fast during the past 100 million years (Myr), adding the equivalent of one human genome every 10 Myr. This massive genome expansion seems to be related to a reduction of PIWI-interacting RNAs and C2H2 zinc-finger and Krüppel-associated box (KRAB)-domain protein genes that suppress TE expansions. Although TE abundance facilitates chromosomal rearrangements, lungfish chromosomes still conservatively reflect the ur-tetrapod karyotype. Neoceratodus' limb-like fins still resemble those of their extinct relatives and remained phenotypically static for about 100 Myr. We show that the secondary loss of limb-like appendages in the Lepidosiren-Protopterus ancestor was probably due to loss of sonic hedgehog limb-specific enhancers.

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades have revealed several disparately related lineages that experienced substantial rearrangements. Most plastomes contain a large inverted repeat and two single-copy regions, and a few dispersed repeats; however, the plastomes of some taxa harbour long repeat sequences (>300 bp). These long repeats make it challenging to assemble complete plastomes using short-read data, leading to misassemblies and consensus sequences with spurious rearrangements. Single-molecule, long-read sequencing has the potential to overcome these challenges, yet there is no consensus on the most effective method for accurately assembling plastomes using long-read data. We generated a pipeline, plastid Genome Assembly Using Long-read data (ptGAUL), to address the problem of plastome assembly using long-read data from Oxford Nanopore Technologies (ONT) or Pacific Biosciences platforms. We demonstrated the efficacy of the ptGAUL pipeline using 16 published long-read data sets. We showed that ptGAUL quickly produces accurate and unbiased assemblies using only ~50× coverage of plastome data. Additionally, we deployed ptGAUL to assemble four new Juncus (Juncaceae) plastomes using ONT long reads. Our results revealed many long repeats and rearrangements in Juncus plastomes compared with basal lineages of Poales. The ptGAUL pipeline is available on GitHub: https://github.com/Bean061/ptgaul.

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR subpathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology and results in sequence insertion without additional breaks or SVs. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Polδ-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.

Anaplastic large cell lymphomas (ALCL) are mature T-cell neoplasms, approximately half of which harbor rearrangements of the ALK gene that confer a good prognosis. Recent studies have demonstrated that a significant proportion of ALK-negative ALCLs demonstrate rearrangements of the IRF4/DUSP22 locus that also are typically associated with a favorable prognosis. ALCL with primary involvement of the central nervous system (CNS) is extremely rare. We report what may be the first case of ALK-negative ALCL with IRF4/DUSP22 rearrangement involving the brain in a 55-year-old man. Magnetic resonance imaging demonstrated signal abnormalities in the periventricular region, corpus callosum and cingulate gyrus. Biopsy revealed a diffuse parenchymal and angiocentric infiltrate of CD30-positive cells that showed IRF4/DUSP22 rearrangement by fluorescence in situ hybridization. We also review the clinical and pathologic features of primary CNS ALK-negative ALCLs in the literature and highlight the need for awareness of this entity to optimize appropriate management.

BackgroundClonal immunoglobulin and T-cell receptor rearrangements serve as tumor-specific markers that have become mainstays of the diagnosis and monitoring of lymphoid malignancy. Next-generation sequencing (NGS) techniques targeting these loci have been successfully applied to lymphoblastic leukemia and multiple myeloma for minimal residual disease detection. However, adoption of NGS for primary diagnosis remains limited.MethodsWe addressed the bioinformatics challenges associated with immune cell sequencing and clone detection by designing a novel web tool, CloneRetriever (CR), which uses machine-learning principles to generate clone classification schemes that are customizable, and can be applied to large datasets. CR has 2 applications-a "validation" mode to derive a clonality classifier, and a "live" mode to screen for clones by applying a validated and/or customized classifier. In this study, CR-generated multiple classifiers using 2 datasets comprising 106 annotated patient samples. A custom classifier was then applied to 36 unannotated samples.ResultsThe optimal classifier for clonality required clonal dominance ≥4.5× above background, read representation ≥8% of all reads, and technical replicate agreement. Depending on the dataset and analysis step, the optimal algorithm yielded sensitivities of 81%-90%, specificities of 97%-100%, areas under the curve of 91%-94%, positive predictive values of 92-100%, and negative predictive values of 88%-98%. Customization of the algorithms yielded 95%-100% concordance with gold-standard clonality determination, including rescue of indeterminate samples. Application to a set of unknowns showed concordance rates of 83%-96%.ConclusionsCR is an out-of-the-box ready and user-friendly software designed to identify clonal rearrangements in large NGS datasets for the diagnosis of lymphoid malignancies.

BackgroundT cell clonality assays in veterinary medicine currently target only the T cell receptor gamma (TRG) locus. Existing assays have suboptimal sensitivity because of insufficient primer coverage of all possible rearrangements.ObjectiveDevelop higher sensitivity clonality assays targeting the TRG, delta (TRD), and beta (TRB) loci in cats.AnimalsCats with histopathologically confirmed lymphoma (n = 89), non-lymphoma (n = 35), and possible hepatic small cell lymphoma (n = 31).MethodsMolecular clonality assay development utilizing our recently reported topology and expressed repertoire data of the T cell receptor loci in cats. Determination of clonality status of lymphoma, non lymphoma, and possible hepatic small cell lymphoma samples, and calculation of assay sensitivity and specificity.ResultsThe new multiplex TRG assay yielded the highest sensitivity (95.5%). All assays yielded 100% specificity except for the new multiplex TRG assay (97.3%). The combination of the new TRG and TRB assays yielded sensitivity of 98.9% and specificity of 97.0%. The new TRG assay detected clonality in 17/31 possible small cell lymphoma livers, whereas an existing TRG assay detected clonality in 6/31 livers.Conclusions and clinical importanceThe assessment of multiple T cell loci compensates for the potential shortcomings of individual assays. Using a combination of molecular clonality assays will increase the overall sensitivity for the diagnosis of T-cell lymphoma in cats, especially intestinal, and hepatic small cell lymphoma. Hepatic small cell lymphomas detected by the new TRG assay utilized rarely expressed V and J genes not recognized by previous assays, likely indicating unique biology of hepatic small cell lymphoma in cats.

Comprehensive characterization of somatic genomic alterations has led to fundamental shifts in our understanding of tumor biology. In clinical practice, these studies can lead to modifications of diagnosis and/or specific treatment implications, fulfilling the promise of personalized medicine. Herein, we describe a 78-yr-old woman under surveillance for long-standing untreated chronic lymphocytic leukemia (CLL). Molecular studies from a peripheral blood specimen revealed a TP53 p.V157F mutation, whereas karyotype and fluorescence in situ hybridization (FISH) identified a 17p deletion, trisomy 12, and no evidence of IGH-CCND1 rearrangement. Positron emission tomography-computed tomography scan identified multistation intra-abdominal lymphadenopathy and a pulmonary nodule, and subsequent pulmonary wedge resection confirmed the presence of a concurrent lung adenocarcinoma. Targeted next-generation sequencing of the lung tumor identified an EGFR in-frame exon 19 deletion, two TP53 mutations (p.P152Q, p.V157F), and, unexpectedly, a IGH-CCND1 rearrangement. Follow-up immunohistochemistry (IHC) studies demonstrated a cyclin D1-positive lymphoid aggregate within the lung adenocarcinoma. The presence of the TP53 p.V157F mutation in the lung resection, detection of an IGH-CCND1 rearrangement, and cyclin D1 positivity by IHC led to revision of the patient's hematologic diagnosis and confirmed the extranodal presence of mantle cell lymphoma within the lung mass, thus representing a "tumor in tumor." Manual review of the sequencing data suggested the IGH-CCND1 rearrangement occurred via an insertional event, whose size precluded detection by original FISH studies. Thus, routine imaging for this patient's known hematologic malignancy led to detection of an unexpected solid tumor, whose subsequent precision medicine studies in the solid tumor redefined the original hematological diagnosis.

Corymbia citriodora is a member of the predominantly Southern Hemisphere Myrtaceae family, which includes the eucalypts (Eucalyptus, Corymbia and Angophora; ~800 species). Corymbia is grown for timber, pulp and paper, and essential oils in Australia, South Africa, Asia, and Brazil, maintaining a high-growth rate under marginal conditions due to drought, poor-quality soil, and biotic stresses. To dissect the genetic basis of these desirable traits, we sequenced and assembled the 408 Mb genome of Corymbia citriodora, anchored into eleven chromosomes. Comparative analysis with Eucalyptus grandis reveals high synteny, although the two diverged approximately 60 million years ago and have different genome sizes (408 vs 641 Mb), with few large intra-chromosomal rearrangements. C. citriodora shares an ancient whole-genome duplication event with E. grandis but has undergone tandem gene family expansions related to terpene biosynthesis, innate pathogen resistance, and leaf wax formation, enabling their successful adaptation to biotic/abiotic stresses and arid conditions of the Australian continent.

Chromoanagenesis is a genomic catastrophe that results in chromosomal shattering and reassembly. These extreme single chromosome events were first identified in cancer, and have since been observed in other systems, but have so far only been formally documented in plants in the context of haploid induction crosses. The frequency, origins, consequences, and evolutionary impact of such major chromosomal remodeling in other situations remain obscure. Here, we demonstrate the occurrence of chromoanagenesis in poplar (Populus sp.) trees produced from gamma-irradiated pollen. Specifically, in this population of siblings carrying indel mutations, two individuals exhibited highly frequent copy number variation (CNV) clustered on a single chromosome, one of the hallmarks of chromoanagenesis. Using short-read sequencing, we confirmed the presence of clustered segmental rearrangement. Independently, we identified and validated novel DNA junctions and confirmed that they were clustered and corresponded to these rearrangements. Our reconstruction of the novel sequences suggests that the chromosomal segments have reorganized randomly to produce a novel rearranged chromosome but that two different mechanisms might be at play. Our results indicate that gamma irradiation can trigger chromoanagenesis, suggesting that this may also occur when natural or induced mutagens cause DNA breaks. We further demonstrate that such events can be tolerated in poplar, and even replicated clonally, providing an attractive system for more in-depth investigations of their consequences.

Corymbia citriodora is a member of the predominantly Southern Hemisphere Myrtaceae family, which includes the eucalypts (Eucalyptus, Corymbia and Angophora; ~800 species). Corymbia is grown for timber, pulp and paper, and essential oils in Australia, South Africa, Asia, and Brazil, maintaining a high-growth rate under marginal conditions due to drought, poor-quality soil, and biotic stresses. To dissect the genetic basis of these desirable traits, we sequenced and assembled the 408 Mb genome of Corymbia citriodora, anchored into eleven chromosomes. Comparative analysis with Eucalyptus grandis reveals high synteny, although the two diverged approximately 60 million years ago and have different genome sizes (408 vs 641 Mb), with few large intra-chromosomal rearrangements. C. citriodora shares an ancient whole-genome duplication event with E. grandis but has undergone tandem gene family expansions related to terpene biosynthesis, innate pathogen resistance, and leaf wax formation, enabling their successful adaptation to biotic/abiotic stresses and arid conditions of the Australian continent.

The human body generates a diverse set of high affinity antibodies, the soluble form of B cell receptors (BCRs), that bind to and neutralize invading pathogens. The natural development of BCRs must be understood in order to design vaccines for highly mutable pathogens such as influenza and HIV. BCR diversity is induced by naturally occurring combinatorial "V(D)J" rearrangement, mutation, and selection processes. Most current methods for BCR sequence analysis focus on separately modeling the above processes. Statistical phylogenetic methods are often used to model the mutational dynamics of BCR sequence data, but these techniques do not consider all the complexities associated with B cell diversification such as the V(D)J rearrangement process. In particular, standard phylogenetic approaches assume the DNA bases of the progenitor (or "naive") sequence arise independently and according to the same distribution, ignoring the complexities of V(D)J rearrangement. In this paper, we introduce a novel approach to Bayesian phylogenetic inference for BCR sequences that is based on a phylogenetic hidden Markov model (phylo-HMM). This technique not only integrates a naive rearrangement model with a phylogenetic model for BCR sequence evolution but also naturally accounts for uncertainty in all unobserved variables, including the phylogenetic tree, via posterior distribution sampling.

PurposeProstate cancer is the second leading cause of male cancer deaths. Castration-resistant prostate cancer (CRPC) is a lethal stage of the disease that emerges when endocrine therapies are no longer effective at suppressing activity of the androgen receptor (AR) transcription factor. The purpose of this study was to identify genomic mechanisms that contribute to the development and progression of CRPC.Experimental designWe used whole-genome and targeted DNA-sequencing approaches to identify mechanisms underlying CRPC in an aggregate cohort of 272 prostate cancer patients. We analyzed structural rearrangements at the genome-wide level and carried out a detailed structural rearrangement analysis of the AR locus. We used genome engineering to perform experimental modeling of AR gene rearrangements and long-read RNA sequencing to analyze effects on expression of AR and truncated AR variants (AR-V).ResultsAR was among the most frequently rearranged genes in CRPC tumors. AR gene rearrangements promoted expression of diverse AR-V species. AR gene rearrangements occurring in the context of AR amplification correlated with AR overexpression. Cell lines with experimentally derived AR gene rearrangements displayed high expression of tumor-specific AR-Vs and were resistant to endocrine therapies, including the AR antagonist enzalutamide.ConclusionsAR gene rearrangements are an important mechanism of resistance to endocrine therapies in CRPC.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare sarcoma subtype characterized by monomorphic epithelioid cells embedded in a densely sclerotic collagenous matrix. The overwhelming majority of tumors arise in soft tissues; however, rare cases have been documented to occur primarily in bone. The hallmarks of soft tissue SEF include MUC4 immunoreactivity and the presence of an EWSR1-CREB3L1 fusion. Rare cases with alternative fusions have also been reported such as EWSR1-CREB3L2 and FUS-CREB3L2 transcripts. The molecular alterations of skeletal SEF have not been well-defined, with only rare cases analyzed to date. In this study we investigated the clinicopathologic and molecular features of seven patients presenting with primary osseous SEF. There were 3 males and 4 females, with a mean age at diagnosis of 38 years. All cases had microscopic features within the histologic spectrum of SEF and showed strong and diffuse MUC4 positivity, while lacking SATB2 expression. However, due to its unusual presentation within bone, four cases were initially misinterpreted as either osteosarcoma, Ewing sarcoma or chondroblastoma. Half of the patients with follow-up data developed metastasis. The cases were tested by targeted RNA sequencing, MSK-IMPACT, and/or fluorescence in situ hybridization, showing EWSR1-CREB3L1 in six cases and EWSR1-CREB3L2 in one case. The fusion transcripts were composed of EWSR1 exon 11 to either exon 6 of CREB3L1 or CREB3L2. In summary, due to their rarity in the bone, skeletal SEF are often misdiagnosed, resulting in inadequate treatment modalities. Similar to their soft tissue counterpart, bone SEF follow an aggressive clinical behavior and show similar EWSR1-CREB3L1/CREB3L2 fusions.

Chromatin is folded into successive layers to organize linear DNA. Genes within the same topologically associating domains (TADs) demonstrate similar expression and histone-modification profiles, and boundaries separating different domains have important roles in reinforcing the stability of these features. Indeed, domain disruptions in human cancers can lead to misregulation of gene expression. However, the frequency of domain disruptions in human cancers remains unclear. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumor types, we analyzed 288,457 somatic structural variations (SVs) to understand the distributions and effects of SVs across TADs. Notably, SVs can lead to the fusion of discrete TADs, and complex rearrangements markedly change chromatin folding maps in the cancer genomes. Notably, only 14% of the boundary deletions resulted in a change in expression in nearby genes of more than twofold.

About half of all cancers have somatic integrations of retrotransposons. Here, to characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 38 histological cancer subtypes within the framework of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. We identified 19,166 somatically acquired retrotransposition events, which affected 35% of samples and spanned a range of event types. Long interspersed nuclear element (LINE-1; L1 hereafter) insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, which sometimes leads to the removal of tumor-suppressor genes, and can induce complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage-fusion-bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications for the development of human tumors.

About half of all cancers have somatic integrations of retrotransposons. Here, to characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 38 histological cancer subtypes within the framework of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. We identified 19,166 somatically acquired retrotransposition events, which affected 35% of samples and spanned a range of event types. Long interspersed nuclear element (LINE-1; L1 hereafter) insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, which sometimes leads to the removal of tumor-suppressor genes, and can induce complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage-fusion-bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications for the development of human tumors.

Chromatin is folded into successive layers to organize linear DNA. Genes within the same topologically associating domains (TADs) demonstrate similar expression and histone-modification profiles, and boundaries separating different domains have important roles in reinforcing the stability of these features. Indeed, domain disruptions in human cancers can lead to misregulation of gene expression. However, the frequency of domain disruptions in human cancers remains unclear. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumor types, we analyzed 288,457 somatic structural variations (SVs) to understand the distributions and effects of SVs across TADs. Notably, SVs can lead to the fusion of discrete TADs, and complex rearrangements markedly change chromatin folding maps in the cancer genomes. Notably, only 14% of the boundary deletions resulted in a change in expression in nearby genes of more than twofold.

A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes1-7. Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types8. Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions-as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2-7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and-in liver cancer-frequently activate the telomerase gene TERT. A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.

A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes1-7. Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types8. Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions-as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2-7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and-in liver cancer-frequently activate the telomerase gene TERT. A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.

Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here, we used a Saccharomyces cerevisiae genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by MRE11, SAE2, SLX1, and YKU80 and large-loop hairpins that were suppressed by YEN1, TEL1, SWR1, and MRC1. Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, POL32-dependent break-induced replication did not appear to be involved forming secondary rearrangements.

The human body generates a diverse set of high affinity antibodies, the soluble form of B cell receptors (BCRs), that bind to and neutralize invading pathogens. The natural development of BCRs must be understood in order to design vaccines for highly mutable pathogens such as influenza and HIV. BCR diversity is induced by naturally occurring combinatorial "V(D)J" rearrangement, mutation, and selection processes. Most current methods for BCR sequence analysis focus on separately modeling the above processes. Statistical phylogenetic methods are often used to model the mutational dynamics of BCR sequence data, but these techniques do not consider all the complexities associated with B cell diversification such as the V(D)J rearrangement process. In particular, standard phylogenetic approaches assume the DNA bases of the progenitor (or "naive") sequence arise independently and according to the same distribution, ignoring the complexities of V(D)J rearrangement. In this paper, we introduce a novel approach to Bayesian phylogenetic inference for BCR sequences that is based on a phylogenetic hidden Markov model (phylo-HMM). This technique not only integrates a naive rearrangement model with a phylogenetic model for BCR sequence evolution but also naturally accounts for uncertainty in all unobserved variables, including the phylogenetic tree, via posterior distribution sampling.

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

Nonself recognition following cell fusion between genetically distinct individuals of the same species in filamentous fungi often results in a programmed cell death (PCD) reaction, where the heterokaryotic fusion cell is compartmentalized and rapidly killed. The allorecognition process plays a key role as a defense mechanism that restricts genome exploitation, resource plundering, and the spread of deleterious senescence plasmids and mycoviruses. Although a number of incompatibility systems have been described that function in mature hyphae, less is known about the PCD pathways in asexual spores, which represent the main infectious unit in various human and plant fungal pathogens. Here, we report the identification of regulator of cell death-1 (rcd-1), a novel allorecognition gene, controlling PCD in germinating asexual spores of Neurospora crassa; rcd-1 is one of the most polymorphic genes in the genomes of wild N. crassa isolates. The coexpression of two antagonistic rcd-1-1 and rcd-1-2 alleles was necessary and sufficient to trigger cell death in fused germlings and in hyphae. Based on analysis of wild populations of N. crassa and N. discreta, rcd-1 alleles appeared to be under balancing selection and associated with trans-species polymorphisms. We shed light on genomic rearrangements that could have led to the emergence of the incompatibility system in Neurospora and show that rcd-1 belongs to a much larger gene family in fungi. Overall, our work contributes toward a better understanding of allorecognition and PCD in an underexplored developmental stage of filamentous fungi.

Nonself recognition following cell fusion between genetically distinct individuals of the same species in filamentous fungi often results in a programmed cell death (PCD) reaction, where the heterokaryotic fusion cell is compartmentalized and rapidly killed. The allorecognition process plays a key role as a defense mechanism that restricts genome exploitation, resource plundering, and the spread of deleterious senescence plasmids and mycoviruses. Although a number of incompatibility systems have been described that function in mature hyphae, less is known about the PCD pathways in asexual spores, which represent the main infectious unit in various human and plant fungal pathogens. Here, we report the identification of regulator of cell death-1 (rcd-1), a novel allorecognition gene, controlling PCD in germinating asexual spores of Neurospora crassa; rcd-1 is one of the most polymorphic genes in the genomes of wild N. crassa isolates. The coexpression of two antagonistic rcd-1-1 and rcd-1-2 alleles was necessary and sufficient to trigger cell death in fused germlings and in hyphae. Based on analysis of wild populations of N. crassa and N. discreta, rcd-1 alleles appeared to be under balancing selection and associated with trans-species polymorphisms. We shed light on genomic rearrangements that could have led to the emergence of the incompatibility system in Neurospora and show that rcd-1 belongs to a much larger gene family in fungi. Overall, our work contributes toward a better understanding of allorecognition and PCD in an underexplored developmental stage of filamentous fungi.

SATB2-associated syndrome (SAS) is an autosomal dominant neurodevelopmental disorder caused by alterations in the SATB2 gene. Here we present a review of published pathogenic variants in the SATB2 gene to date and report 38 novel alterations found in 57 additional previously unreported individuals. Overall, we present a compilation of 120 unique variants identified in 155 unrelated families ranging from single nucleotide coding variants to genomic rearrangements distributed throughout the entire coding region of SATB2. Single nucleotide variants predicted to result in the occurrence of a premature stop codon were the most commonly seen (51/120 = 42.5%) followed by missense variants (31/120 = 25.8%). We review the rather limited functional characterization of pathogenic variants and discuss current understanding of the consequences of the different molecular alterations. We present an expansive phenotypic review along with novel genotype-phenotype correlations. Lastly, we discuss current knowledge of animal models and present future prospects. This review should help provide better guidance for the care of individuals diagnosed with SAS.

Well-preserved mRNA in circulating tumor cells (CTCs) offers an ideal material for conducting molecular profiling of tumors, thereby providing a noninvasive diagnostic solution for guiding treatment intervention and monitoring disease progression. However, it is technically challenging to purify CTCs while retaining high-quality mRNA.Here, we demonstrate a covalent chemistry-based nanostructured silicon substrate ("Click Chip") for CTC purification that leverages bioorthogonal ligation-mediated CTC capture and disulfide cleavage-driven CTC release. This platform is ideal for CTC mRNA assays because of its efficient, specific, and rapid purification of pooled CTCs, enabling downstream molecular quantification using reverse transcription Droplet Digital polymerase chain reaction. Rearrangements of ALK/ROS1 were quantified using CTC mRNA and matched with those identified in biopsy specimens from 12 patients with late-stage non-small cell lung cancer. Moreover, CTC counts and copy numbers of ALK/ROS1 rearrangements could be used together for evaluating treatment responses and disease progression.

Well-preserved mRNA in circulating tumor cells (CTCs) offers an ideal material for conducting molecular profiling of tumors, thereby providing a noninvasive diagnostic solution for guiding treatment intervention and monitoring disease progression. However, it is technically challenging to purify CTCs while retaining high-quality mRNA.Here, we demonstrate a covalent chemistry-based nanostructured silicon substrate ("Click Chip") for CTC purification that leverages bioorthogonal ligation-mediated CTC capture and disulfide cleavage-driven CTC release. This platform is ideal for CTC mRNA assays because of its efficient, specific, and rapid purification of pooled CTCs, enabling downstream molecular quantification using reverse transcription Droplet Digital polymerase chain reaction. Rearrangements of ALK/ROS1 were quantified using CTC mRNA and matched with those identified in biopsy specimens from 12 patients with late-stage non-small cell lung cancer. Moreover, CTC counts and copy numbers of ALK/ROS1 rearrangements could be used together for evaluating treatment responses and disease progression.

Tumor-derived extracellular vesicles (EVs) present in bodily fluids are emerging liquid biopsy markers for non-invasive cancer diagnosis and treatment monitoring. Because the majority of EVs in circulation are not of tumor origin, it is critical to develop new platforms capable of enriching tumor-derived EVs from the blood. Herein, we introduce a biostructure-inspired NanoVilli Chip, capable of highly efficient and reproducible immunoaffinity capture of tumor-derived EVs from blood plasma samples. Anti-EpCAM-grafted silicon nanowire arrays were engineered to mimic the distinctive structures of intestinal microvilli, dramatically increasing surface area and enhancing tumor-derived EV capture. RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment responses and disease progression in non-small cell lung cancer patients.

The immunoglobulin heavy variable (IGHV) and T cell beta variable (TRBV) loci are among the most complex and variable regions in the human genome. Generated through a process of gene duplication/deletion and diversification, these loci can vary extensively between individuals in copy number and contain genes that are highly similar, making their analysis technically challenging. Here, we present a comprehensive study of the functional gene segments in the IGHV and TRBV loci, quantifying their copy number and single-nucleotide variation in a globally diverse sample of 109 (IGHV) and 286 (TRBV) humans from over a 100 populations. We find that the IGHV and TRBV gene families exhibit starkly different patterns of variation. In addition to providing insight into the different evolutionary paths of the IGHV and TRBV loci, our results are also important to the adaptive immune repertoire sequencing community, where the lack of frequencies of common alleles and copy number variants is hampering existing analytical pipelines.

Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed CEN-SELECT, to systematically interrogate the structural landscape of mis-segregated chromosomes. We show that single-chromosome mis-segregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that mis-segregated chromosomes exhibit 120-fold-higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.

Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed CEN-SELECT, to systematically interrogate the structural landscape of mis-segregated chromosomes. We show that single-chromosome mis-segregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that mis-segregated chromosomes exhibit 120-fold-higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.

The immunoglobulin heavy variable (IGHV) and T cell beta variable (TRBV) loci are among the most complex and variable regions in the human genome. Generated through a process of gene duplication/deletion and diversification, these loci can vary extensively between individuals in copy number and contain genes that are highly similar, making their analysis technically challenging. Here, we present a comprehensive study of the functional gene segments in the IGHV and TRBV loci, quantifying their copy number and single-nucleotide variation in a globally diverse sample of 109 (IGHV) and 286 (TRBV) humans from over a 100 populations. We find that the IGHV and TRBV gene families exhibit starkly different patterns of variation. In addition to providing insight into the different evolutionary paths of the IGHV and TRBV loci, our results are also important to the adaptive immune repertoire sequencing community, where the lack of frequencies of common alleles and copy number variants is hampering existing analytical pipelines.

IntroductionGenomic profiling informs selection of matched targeted therapies as part of routine clinical care in NSCLC. Tissue biopsy is the criterion standard; however, genomic profiling of blood-derived circulating tumor DNA (ctDNA) has emerged as a minimally invasive alternative.MethodsHybrid capture-based genomic profiling of 62 genes was performed on blood-based ctDNA from 1552 patients with NSCLC.ResultsEvidence of ctDNA was detected in 80% of samples, and in 86% of these cases, at least one reportable genomic alteration (GA) was detected. Frequently altered genes were tumor protein p53 gene (TP53) (59%), EGFR (25%), and KRAS (17%). Comparative analysis with a tissue genomic database (N = 21,500) showed similar frequencies of GAs per gene, although KRAS mutation and EGFR T790M were more frequent in tissue and ctDNA, respectively (both p < 0.0001), likely reflecting the use of liquid versus tissue biopsy after relapse during targeted therapy. In temporally matched ctDNA and tissue samples from 33 patients with evidence of ctDNA in their blood, 64% of GAs detected in tissue were also detected in ctDNA, including 78% of short variants (58 of 74) and 100% of rearrangements (four of four), but only 16% of amplifications (four of 25).ConclusionsGenomic profiling of ctDNA detected clinically relevant GAs in a significant subset of NSCLC cases. Most alterations detected in matched tissue were also detected in ctDNA. These results suggest the utility of ctDNA testing in advanced NSCLC as a complementary approach to tissue testing. Blood-based ctDNA testing may be particularly useful at the time of progression during targeted therapy.

IntroductionGenomic profiling informs selection of matched targeted therapies as part of routine clinical care in NSCLC. Tissue biopsy is the criterion standard; however, genomic profiling of blood-derived circulating tumor DNA (ctDNA) has emerged as a minimally invasive alternative.MethodsHybrid capture-based genomic profiling of 62 genes was performed on blood-based ctDNA from 1552 patients with NSCLC.ResultsEvidence of ctDNA was detected in 80% of samples, and in 86% of these cases, at least one reportable genomic alteration (GA) was detected. Frequently altered genes were tumor protein p53 gene (TP53) (59%), EGFR (25%), and KRAS (17%). Comparative analysis with a tissue genomic database (N = 21,500) showed similar frequencies of GAs per gene, although KRAS mutation and EGFR T790M were more frequent in tissue and ctDNA, respectively (both p < 0.0001), likely reflecting the use of liquid versus tissue biopsy after relapse during targeted therapy. In temporally matched ctDNA and tissue samples from 33 patients with evidence of ctDNA in their blood, 64% of GAs detected in tissue were also detected in ctDNA, including 78% of short variants (58 of 74) and 100% of rearrangements (four of four), but only 16% of amplifications (four of 25).ConclusionsGenomic profiling of ctDNA detected clinically relevant GAs in a significant subset of NSCLC cases. Most alterations detected in matched tissue were also detected in ctDNA. These results suggest the utility of ctDNA testing in advanced NSCLC as a complementary approach to tissue testing. Blood-based ctDNA testing may be particularly useful at the time of progression during targeted therapy.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy.

Sequencing data have been instrumental in identifying oncogenic drivers in prostatic carcinoma and highlighting biomarkers that define aggressive disease. A review of a series of 30 primary and metastatic prostatic carcinomas clinically sequenced at our cancer genomics laboratory utilizing a targeted gene panel identified recurrent structural variants in the TP53 gene. These structural variants were found in 27% of all sequenced cases and represented 36% of the cases with metastatic disease. TP53 structural rearrangements have been previously reported in a significant subset of osteosarcomas, where they result in loss of p53 protein expression by immunohistochemistry. Similarly, in our prostate cases with TP53 structural rearrangements for which tissue was available for testing, we find loss of p53 protein expression by immunohistochemistry. In the eight TP53-rearranged cases, concurrent PTEN loss was identified in 4 cases, TMPRSS2-ERG fusion in 5 cases, and AR and FOXA1 amplification in 1 case each. Our results from this small case series suggest that TP53 rearrangements with loss of expression represent a frequent alternative mechanism of inactivation of this key tumor suppressor gene with potential utility as a marker of aggressive disease. Recognition of this TP53 rearrangement pathway is essential to accurately identify prostatic carcinomas with loss of TP53 function.

Sequencing data have been instrumental in identifying oncogenic drivers in prostatic carcinoma and highlighting biomarkers that define aggressive disease. A review of a series of 30 primary and metastatic prostatic carcinomas clinically sequenced at our cancer genomics laboratory utilizing a targeted gene panel identified recurrent structural variants in the TP53 gene. These structural variants were found in 27% of all sequenced cases and represented 36% of the cases with metastatic disease. TP53 structural rearrangements have been previously reported in a significant subset of osteosarcomas, where they result in loss of p53 protein expression by immunohistochemistry. Similarly, in our prostate cases with TP53 structural rearrangements for which tissue was available for testing, we find loss of p53 protein expression by immunohistochemistry. In the eight TP53-rearranged cases, concurrent PTEN loss was identified in 4 cases, TMPRSS2-ERG fusion in 5 cases, and AR and FOXA1 amplification in 1 case each. Our results from this small case series suggest that TP53 rearrangements with loss of expression represent a frequent alternative mechanism of inactivation of this key tumor suppressor gene with potential utility as a marker of aggressive disease. Recognition of this TP53 rearrangement pathway is essential to accurately identify prostatic carcinomas with loss of TP53 function.

Ectomesenchymal chondromyxoid tumor is a rare and benign neoplasm with a predilection for the anterior dorsal tongue. Despite morphologic heterogeneity, most cases are characterized by a proliferation of bland spindle cells with a distinctive reticular growth pattern and myxoid stroma. The immunophenotype of these neoplasms is likewise variable; most cases express glial fibrillary acid protein and S100 protein, with inconsistent reports of keratin and myoid marker expression. The molecular pathogenesis is poorly understood; however, a subset of cases has been reported to harbor EWSR1 gene rearrangement. Following identification of an RREB1-MKL2 fusion gene by RNA Sequencing in an index patient, a retrospective review of additional cases of ectomesenchymal chondromyxoid tumors was performed to better characterize the clinical, immunohistochemical, and molecular attributes of this neoplasm. A total of 21 cases were included in this series. A marked predisposition for the dorsal tongue was confirmed. Most cases conformed to prior morphologic descriptions; however, hypercellularity, hyalinized stroma, and necrosis were rare attributes not previously emphasized. The neoplastic cells frequently coexpressed glial fibrillary acid protein, S100 protein, keratin, smooth muscle actin, and/or desmin; a single case was found to contain significant myogenin expression. An RREB1-MKL2 fusion product was identified in 19 tumors (90%), a single tumor (5%) had an EWSR1-CREM fusion product, and the remaining case lacked any known fusion gene by RNA Sequencing. The latter 2 cases subtly differed morphologically from many in the cohort. This series illustrates that recurrent RREB1-MKL2 fusions occur in most, perhaps all, cases of ectomesenchymal chondromyxoid tumor.

Genome instability is associated with tumorigenesis. Here, we identify a role for the histone Htz1, which is deposited by the Swr1 chromatin-remodeling complex (SWR-C), in preventing genome instability in the absence of the replication fork/replication checkpoint proteins Mrc1, Csm3, or Tof1. When combined with deletion of SWR1 or HTZ1, deletion of MRC1, CSM3, or TOF1 or a replication-defective mrc1 mutation causes synergistic increases in gross chromosomal rearrangement (GCR) rates, accumulation of a broad spectrum of GCRs, and hypersensitivity to replication stress. The double mutants have severe replication defects and accumulate aberrant replication intermediates. None of the individual mutations cause large increases in GCR rates; however, defects in MRC1, CSM3 or TOF1 cause activation of the DNA damage checkpoint and replication defects. We propose a model in which Htz1 deposition and retention in chromatin prevents transiently stalled replication forks that occur in mrc1, tof1, or csm3 mutants from being converted to DNA double-strand breaks that trigger genome instability.

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease.

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged, although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available, with nearly 20,000 variants present, most of which were missed by short-read sequencing. Surrounding the important ERBB2 oncogene (also known as HER2), we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the genome and sheds new light on the complex mechanisms involved in cancer genome evolution.

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease.

Separate germline and somatic genomes are found in numerous lineages across the eukaryotic tree of life, often separated into distinct tissues (e.g., in plants, animals, and fungi) or distinct nuclei sharing a common cytoplasm (e.g., in ciliates and some foraminifera). In ciliates, germline-limited (i.e., micronuclear-specific) DNA is eliminated during the development of a new somatic (i.e., macronuclear) genome in a process that is tightly linked to large-scale genome rearrangements, such as deletions and reordering of protein-coding sequences. Most studies of germline genome architecture in ciliates have focused on the model ciliates Oxytricha trifallax, Paramecium tetraurelia, and Tetrahymena thermophila, for which the complete germline genome sequences are known. Outside of these model taxa, only a few dozen germline loci have been characterized from a limited number of cultivable species, which is likely due to difficulties in obtaining sufficient quantities of "purified" germline DNA in these taxa. Combining single-cell transcriptomics and genomics, we have overcome these limitations and provide the first insights into the structure of the germline genome of the ciliate Chilodonella uncinata, a member of the understudied class Phyllopharyngea Our analyses reveal the following: (i) large gene families contain a disproportionate number of genes from scrambled germline loci; (ii) germline-soma boundaries in the germline genome are demarcated by substantial shifts in GC content; (iii) single-cell omics techniques provide large-scale quality germline genome data with limited effort, at least for ciliates with extensively fragmented somatic genomes. Our approach provides an efficient means to understand better the evolution of genome rearrangements between germline and soma in ciliates.IMPORTANCE Our understanding of the distinctions between germline and somatic genomes in ciliates has largely relied on studies of a few model genera (e.g., Oxytricha, Paramecium, Tetrahymena). We have used single-cell omics to explore germline-soma distinctions in the ciliate Chilodonella uncinata, which likely diverged from the better-studied ciliates ~700 million years ago. The analyses presented here indicate that developmentally regulated genome rearrangements between germline and soma are demarcated by rapid transitions in local GC composition and lead to diversification of protein families. The approaches used here provide the basis for future work aimed at discerning the evolutionary impacts of germline-soma distinctions among diverse ciliates.

The mechanisms that mediate durable protection against Staphylococcus aureus skin reinfections are unclear, as recurrences are common despite high antibody titers and memory T cells. Here, we developed a mouse model of S. aureus skin reinfection to investigate protective memory responses. In contrast with WT mice, IL-1β-deficient mice exhibited poor neutrophil recruitment and bacterial clearance during primary infection that was rescued during secondary S. aureus challenge. The γδ T cells from skin-draining LNs utilized compensatory T cell-intrinsic TLR2/MyD88 signaling to mediate rescue by trafficking and producing TNF and IFN-γ, which restored neutrophil recruitment and promoted bacterial clearance. RNA-sequencing (RNA-seq) of the LNs revealed a clonotypic S. aureus-induced γδ T cell expansion with a complementarity-determining region 3 (CDR3) aa sequence identical to that of invariant Vγ5+ dendritic epidermal T cells. However, this T cell receptor γ (TRG) aa sequence of the dominant CDR3 sequence was generated from multiple gene rearrangements of TRGV5 and TRGV6, indicating clonotypic expansion. TNF- and IFN-γ-producing γδ T cells were also expanded in peripheral blood of IRAK4-deficient humans no longer predisposed to S. aureus skin infections. Thus, clonally expanded γδ T cells represent a mechanism for long-lasting immunity against recurrent S. aureus skin infections.