Genome-wide reconstruction of complex structural variants using read clouds.

In read cloud approaches, microfluidic partitioning of long genomic DNA fragments and barcoding of shorter fragments derived from these fragments retains long-range information in short sequencing reads. This combination of short reads with long-range information represents a powerful alternative to single-molecule long-read sequencing. We develop Genome-wide Reconstruction of Complex Structural Variants (GROC-SVs) for SV detection and assembly from read cloud data and apply this method to Illumina-sequenced 10x Genomics sarcoma and breast cancer data sets. Compared with short-fragment sequencing, GROC-SVs substantially improves the specificity of breakpoint detection at comparable sensitivity. This approach also performs sequence assembly across multiple breakpoints simultaneously, enabling the reconstruction of events exhibiting remarkable complexity. We show that chromothriptic rearrangements occurred before copy number amplifications, and that rates of single-nucleotide variants and SVs are not correlated. Our results support the use of read cloud approaches to advance the characterization of large and complex structural variation.

Read clouds uncover variation in complex regions of the human genome.

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies.

Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California.

Strain differentiating marker profiles of citrus tristeza virus (CTV) isolates from California have shown the presence of multiple genotypes. To better define the genetic diversity involved, full-length genome sequences from four California CTV isolates were determined by small-interfering RNA sequencing. Phylogenetic analysis and nucleotide sequence comparisons differentiated these isolates into the genotypes VT (CA-VT-AT39), T30 (CA-T30-AT4), and a new strain called S1 (CA-S1-L and CA-S1-L65). S1 isolates had three common recombination events within portions of genes from VT, T36 and RB strains and were transmissible by Aphis gossypii. Virus indexing showed that CA-VT-AT39 could be classified as a severe strain, whereas CA-T30-AT4, CA-S1-L and CA-S1-L65 were mild. CA-VT-AT39, CA-S1-L, and CA-S1-L65 reacted with monoclonal antibody MCA13, whereas CA-T30-AT4 did not. RT-PCR and RT-qPCR detection assays for the S1 strain were developed and used to screen MCA13-reactive isolates in a CTV collection from central California collected from 1968 to 2011. Forty-two isolates were found to contain the S1 strain, alone or in combinations with other genotypes. BLAST and phylogenetic analysis of the S1 p25 gene region with other extant CTV sequences from the NCBI database suggested that putative S1-like isolates might occur elsewhere (e.g., China, South Korea, Turkey, Bosnia and Croatia). This information is important for CTV evolution, detection of specific strains, and cross-protection.

Discovery of recurrent structural variants in nasopharyngeal carcinoma.

We present the discovery of genes recurrently involved in structural variation in nasopharyngeal carcinoma (NPC) and the identification of a novel type of somatic structural variant. We identified the variants with high complexity mate-pair libraries and a novel computational algorithm specifically designed for tumor-normal comparisons, SMASH. SMASH combines signals from split reads and mate-pair discordance to detect somatic structural variants. We demonstrate a >90% validation rate and a breakpoint reconstruction accuracy of 3 bp by Sanger sequencing. Our approach identified three in-frame gene fusions (YAP1-MAML2, PTPLB-RSRC1, and SP3-PTK2) that had strong levels of expression in corresponding NPC tissues. We found two cases of a novel type of structural variant, which we call "coupled inversion," one of which produced the YAP1-MAML2 fusion. To investigate whether the identified fusion genes are recurrent, we performed fluorescent in situ hybridization (FISH) to screen 196 independent NPC cases. We observed recurrent rearrangements of MAML2 (three cases), PTK2 (six cases), and SP3 (two cases), corresponding to a combined rate of structural variation recurrence of 6% among tested NPC tissues.

Genome evolution during progression to breast cancer.

Cancer evolution involves cycles of genomic damage, epigenetic deregulation, and increased cellular proliferation that eventually culminate in the carcinoma phenotype. Early neoplasias, which are often found concurrently with carcinomas and are histologically distinguishable from normal breast tissue, are less advanced in phenotype than carcinomas and are thought to represent precursor stages. To elucidate their role in cancer evolution we performed comparative whole-genome sequencing of early neoplasias, matched normal tissue, and carcinomas from six patients, for a total of 31 samples. By using somatic mutations as lineage markers we built trees that relate the tissue samples within each patient. On the basis of these lineage trees we inferred the order, timing, and rates of genomic events. In four out of six cases, an early neoplasia and the carcinoma share a mutated common ancestor with recurring aneuploidies, and in all six cases evolution accelerated in the carcinoma lineage. Transition spectra of somatic mutations are stable and consistent across cases, suggesting that accumulation of somatic mutations is a result of increased ancestral cell division rather than specific mutational mechanisms. In contrast to highly advanced tumors that are the focus of much of the current cancer genome sequencing, neither the early neoplasia genomes nor the carcinomas are enriched with potentially functional somatic point mutations. Aneuploidies that occur in common ancestors of neoplastic and tumor cells are the earliest events that affect a large number of genes and may predispose breast tissue to eventual development of invasive carcinoma.

Aquila enables reference-assisted diploid personal genome assembly and comprehensive variant detection based on linked reads.

We introduce Aquila, a new approach to variant discovery in personal genomes, which is critical for uncovering the genetic contributions to health and disease. Aquila uses a reference sequence and linked-read data to generate a high quality diploid genome assembly, from which it then comprehensively detects and phases personal genetic variation. The contigs of the assemblies from our libraries cover >95% of the human reference genome, with over 98% of that in a diploid state. Thus, the assemblies support detection and accurate genotyping of the most prevalent types of human genetic variation, including single nucleotide polymorphisms (SNPs), small insertions and deletions (small indels), and structural variants (SVs), in all but the most difficult regions. All heterozygous variants are phased in blocks that can approach arm-level length. The final output of Aquila is a diploid and phased personal genome sequence, and a phased Variant Call Format (VCF) file that also contains homozygous and a few unphased heterozygous variants. Aquila represents a cost-effective approach that can be applied to cohorts for variation discovery or association studies, or to single individuals with rare phenotypes that could be caused by SVs or compound heterozygosity.

De novo diploid genome assembly for genome-wide structural variant detection.

Detection of structural variants (SVs) on the basis of read alignment to a reference genome remains a difficult problem. De novo assembly, traditionally used to generate reference genomes, offers an alternative for SV detection. However, it has not been applied broadly to human genomes because of fundamental limitations of short-fragment approaches and high cost of long-read technologies. We here show that 10× linked-read sequencing supports accurate SV detection. We examined variants in six de novo 10× assemblies with diverse experimental parameters from two commonly used human cell lines: NA12878 and NA24385. The assemblies are effective for detecting mid-size SVs, which were discovered by simple pairwise alignment of the assemblies' contigs to the reference (hg38). Our study also shows that the base-pair level SV breakpoint accuracy is high, with a majority of SVs having precisely correct sizes and breakpoints. Setting the ancestral state of SV loci by comparing to ape orthologs allows inference of the actual molecular mechanism (insertion or deletion) causing the mutation. In about half of cases, the mechanism is the opposite of the reference-based call. We uncover 214 SVs that may have been maintained as polymorphisms in the human lineage since before our divergence from chimp. Overall, we show that de novo assembly of 10× linked-read data can achieve cost-effective SV detection for personal genomes.

Strain-resolved microbiome sequencing reveals mobile elements that drive bacterial competition on a clinical timescale.

BACKGROUND: Populations of closely related microbial strains can be simultaneously present in bacterial communities such as the human gut microbiome. We recently developed a de novo genome assembly approach that uses read cloud sequencing to provide more complete microbial genome drafts, enabling precise differentiation and tracking of strain-level dynamics across metagenomic samples. In this case study, we present a proof-of-concept using read cloud sequencing to describe bacterial strain diversity in the gut microbiome of one hematopoietic cell transplantation patient over a 2-month time course and highlight temporal strain variation of gut microbes during therapy. The treatment was accompanied by diet changes and administration of multiple immunosuppressants and antimicrobials. METHODS: We conducted short-read and read cloud metagenomic sequencing of DNA extracted from four longitudinal stool samples collected during the course of treatment of one hematopoietic cell transplantation (HCT) patient. After applying read cloud metagenomic assembly to discover strain-level sequence variants in these complex microbiome samples, we performed metatranscriptomic analysis to investigate differential expression of antibiotic resistance genes. Finally, we validated predictions from the genomic and metatranscriptomic findings through in vitro antibiotic susceptibility testing and whole genome sequencing of isolates derived from the patient stool samples. RESULTS: During the 56-day longitudinal time course that was studied, the patient's microbiome was profoundly disrupted and eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allowed us to identify differences in substrain populations over time. Based on this, we predicted that particular mobile element integrations likely resulted in increased antibiotic resistance, which we further supported using in vitro antibiotic susceptibility testing. CONCLUSIONS: We find read cloud assembly to be useful in identifying key structural genomic strain variants within a metagenomic sample. These strains have fluctuating relative abundance over relatively short time periods in human microbiomes. We also find specific structural genomic variations that are associated with increased antibiotic resistance over the course of clinical treatment.

Assessment of human diploid genome assembly with 10x Linked-Reads data.

BACKGROUND: Producing cost-effective haplotype-resolved personal genomes remains challenging. 10x Linked-Read sequencing, with its high base quality and long-range information, has been demonstrated to facilitate de novo assembly of human genomes and variant detection. In this study, we investigate in depth how the parameter space of 10x library preparation and sequencing affects assembly quality, on the basis of both simulated and real libraries. RESULTS: We prepared and sequenced eight 10x libraries with a diverse set of parameters from standard cell lines NA12878 and NA24385 and performed whole-genome assembly on the data. We also developed the simulator LRTK-SIM to follow the workflow of 10x data generation and produce realistic simulated Linked-Read data sets. We found that assembly quality could be improved by increasing the total sequencing coverage (C) and keeping physical coverage of DNA fragments (CF) or read coverage per fragment (CR) within broad ranges. The optimal physical coverage was between 332× and 823× and assembly quality worsened if it increased to >1,000× for a given C. Long DNA fragments could significantly extend phase blocks but decreased contig contiguity. The optimal length-weighted fragment length (W${\mu _{FL}}$) was ∼50-150 kb. When broadly optimal parameters were used for library preparation and sequencing, ∼80% of the genome was assembled in a diploid state. CONCLUSIONS: The Linked-Read libraries we generated and the parameter space we identified provide theoretical considerations and practical guidelines for personal genome assemblies based on 10x Linked-Read sequencing.

Comprehensive genomic characterization of breast tumors with BRCA1 and BRCA2 mutations.

BACKGROUND: Germline mutations in the BRCA1 and BRCA2 genes predispose carriers to breast and ovarian cancer, and there remains a need to identify the specific genomic mechanisms by which cancer evolves in these patients. Here we present a systematic genomic analysis of breast tumors with BRCA1 and BRCA2 mutations. METHODS: We analyzed genomic data from breast tumors, with a focus on comparing tumors with BRCA1/BRCA2 gene mutations with common classes of sporadic breast tumors. RESULTS: We identify differences between BRCA-mutated and sporadic breast tumors in patterns of point mutation, DNA methylation and structural variation. We show that structural variation disproportionately affects tumor suppressor genes and identify specific driver gene candidates that are enriched for structural variation. CONCLUSIONS: Compared to sporadic tumors, BRCA-mutated breast tumors show signals of reduced DNA methylation, more ancestral cell divisions, and elevated rates of structural variation that tend to disrupt highly expressed protein-coding genes and known tumor suppressors. Our analysis suggests that BRCA-mutated tumors are more aggressive than sporadic breast cancers because loss of the BRCA pathway causes multiple processes of mutagenesis and gene dysregulation.

High-quality genome sequences of uncultured microbes by assembly of read clouds.

Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality microbial genome drafts without isolation and culture remains difficult. Here, we present an application of read clouds, short-read sequences tagged with long-range information, to microbiome samples. We present Athena, a de novo assembler that uses read clouds to improve metagenomic assemblies. We applied this approach to sequence stool samples from two healthy individuals and compared it with existing short-read and synthetic long-read metagenomic sequencing techniques. Read-cloud metagenomic sequencing and Athena assembly produced the most comprehensive individual genome drafts with high contiguity (>200-kb N50, fewer than ten contigs), even for bacteria with relatively low (20×) raw short-read-sequence coverage. We also sequenced a complex marine-sediment sample and generated 24 intermediate-quality genome drafts (>70% complete, <10% contaminated), nine of which were complete (>90% complete, <5% contaminated). Our approach allows for culture-free generation of high-quality microbial genome drafts by using a single shotgun experiment.

CBP Modulates Sensitivity to Dasatinib in Pre-BCR+ Acute Lymphoblastic Leukemia.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional coactivator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due, in part, to upregulation of alternative pathways including WNT through a mechanism, suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or ß-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small-molecule inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL.Significance: These findings reveal mechanisms that modulate sensitivity to dasatinib and suggest therapeutic strategies to improve the outcome of patients with acute lymphoblastic leukemia.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6497/F1.large.jpg Cancer Res; 78(22); 6497-508. ©2018 AACR.

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.

Extensive sequencing of seven human genomes to characterize benchmark reference materials.

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

Cell-lineage heterogeneity and driver mutation recurrence in pre-invasive breast neoplasia.

BACKGROUND: All cells in an individual are related to one another by a bifurcating lineage tree, in which each node is an ancestral cell that divided into two, each branch connects two nodes, and the root is the zygote. When a somatic mutation occurs in an ancestral cell, all its descendants carry the mutation, which can then serve as a lineage marker for the phylogenetic reconstruction of tumor progression. Using this concept, we investigate cell lineage relationships and genetic heterogeneity of pre-invasive neoplasias compared to invasive carcinomas. METHODS: We deeply sequenced over a thousand phylogenetically informative somatic variants in 66 morphologically independent samples from six patients that represent a spectrum of normal, early neoplasia, carcinoma in situ, and invasive carcinoma. For each patient, we obtained a highly resolved lineage tree that establishes the phylogenetic relationships among the pre-invasive lesions and with the invasive carcinoma. RESULTS: The trees reveal lineage heterogeneity of pre-invasive lesions, both within the same lesion, and between histologically similar ones. On the basis of the lineage trees, we identified a large number of independent recurrences of PIK3CA H1047 mutations in separate lesions in four of the six patients, often separate from the diagnostic carcinoma. CONCLUSIONS: Our analyses demonstrate that multi-sample phylogenetic inference provides insights on the origin of driver mutations, lineage heterogeneity of neoplastic proliferations, and the relationship of genomically aberrant neoplasias with the primary tumors. PIK3CA driver mutations may be comparatively benign inducers of cellular proliferation.

The H3K4-Methyl Epigenome Regulates Leukemia Stem Cell Oncogenic Potential.

The genetic programs that maintain leukemia stem cell (LSC) self-renewal and oncogenic potential have been well defined; however, the comprehensive epigenetic landscape that sustains LSC cellular identity and functionality is less well established. We report that LSCs in MLL-associated leukemia reside in an epigenetic state of relative genome-wide high-level H3K4me3 and low-level H3K79me2. LSC differentiation is associated with reversal of these broad epigenetic profiles, with concomitant downregulation of crucial MLL target genes and the LSC maintenance transcriptional program that is driven by the loss of H3K4me3, but not H3K79me2. The H3K4-specific demethylase KDM5B negatively regulates leukemogenesis in murine and human MLL-rearranged AML cells, demonstrating a crucial role for the H3K4 global methylome in determining LSC fate.

3'-end sequencing for expression quantification (3SEQ) from archival tumor samples.

Gene expression microarrays are the most widely used technique for genome-wide expression profiling. However, microarrays do not perform well on formalin fixed paraffin embedded tissue (FFPET). Consequently, microarrays cannot be effectively utilized to perform gene expression profiling on the vast majority of archival tumor samples. To address this limitation of gene expression microarrays, we designed a novel procedure (3'-end sequencing for expression quantification (3SEQ)) for gene expression profiling from FFPET using next-generation sequencing. We performed gene expression profiling by 3SEQ and microarray on both frozen tissue and FFPET from two soft tissue tumors (desmoid type fibromatosis (DTF) and solitary fibrous tumor (SFT)) (total n = 23 samples, which were each profiled by at least one of the four platform-tissue preparation combinations). Analysis of 3SEQ data revealed many genes differentially expressed between the tumor types (FDR<0.01) on both the frozen tissue (approximately 9.6K genes) and FFPET (approximately 8.1K genes). Analysis of microarray data from frozen tissue revealed fewer differentially expressed genes (approximately 4.64K), and analysis of microarray data on FFPET revealed very few (69) differentially expressed genes. Functional gene set analysis of 3SEQ data from both frozen tissue and FFPET identified biological pathways known to be important in DTF and SFT pathogenesis and suggested several additional candidate oncogenic pathways in these tumors. These findings demonstrate that 3SEQ is an effective technique for gene expression profiling from archival tumor samples and may facilitate significant advances in translational cancer research.

Three discontinuous loop nucleotides in the 3' terminal stem-loop are required for Red clover necrotic mosaic virus RNA-2 replication.

The genome of Red clover necrotic mosaic virus (RCNMV) consists of positive-sense, single-stranded RNA-1 and RNA-2. The 29 nucleotides at the 3' termini of both RNAs are nearly identical and are predicted to form a stable stem-loop (SL) structure, which is required for RCNMV RNA replication. Here we performed a systematic mutagenesis of the RNA-2 3' SL to identify the nucleotides critical for replication. Infectivity and RNA replication assays indicated that the secondary structure of the 3' SL and its loop sequence UAUAA were required for RNA replication. Single-nucleotide substitution analyses of the loop further pinpointed three discontinuous nucleotides (L1U, L2A, and L4A) that were vital for RNA replication. A 3-D model of the 3' SL predicted the existence of a pocket formed by these three nucleotides that could be involved in RNA-protein interaction. The functional groups of the bases participating in this interaction at these positions are discussed.

Persistent infection and promiscuous recombination of multiple genotypes of an RNA virus within a single host generate extensive diversity.

Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses.