Random forest classifiers trained on simulated data enable accurate short read-based genotyping of structural variants in the alpha globin region at Chr16p13.3.

In regions where reads don't align well to a reference, it is generally difficult to characterize structural variation using short read sequencing. Here, we utilize machine learning classifiers and short sequence reads to genotype structural variants in the alpha globin locus on chromosome 16, a medically-relevant region that is challenging to genotype in individuals. Using models trained only with simulated data, we accurately genotype two hard-to-distinguish deletions in two separate human cohorts. Furthermore, population allele frequencies produced by our methods across a wide set of ancestries agree more closely with previously-determined frequencies than those obtained using currently available genotyping software.

The complete sequence of a human Y chromosome.

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure that includes long palindromes, tandem repeats and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4,5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, showing the complete ampliconic structures of gene families TSPY, DAZ and RBMY; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a previous assembly of the CHM13 genome4 and mapped available population variation, clinical variants and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

Long-read mapping to repetitive reference sequences using Winnowmap2.

Approximately 5-10% of the human genome remains inaccessible due to the presence of repetitive sequences such as segmental duplications and tandem repeat arrays. We show that existing long-read mappers often yield incorrect alignments and variant calls within long, near-identical repeats, as they remain vulnerable to allelic bias. In the presence of a nonreference allele within a repeat, a read sampled from that region could be mapped to an incorrect repeat copy. To address this limitation, we developed a new long-read mapping method, Winnowmap2, by using minimal confidently alignable substrings. Winnowmap2 computes each read mapping through a collection of confident subalignments. This approach is more tolerant of structural variation and more sensitive to paralog-specific variants within repeats. Our experiments highlight that Winnowmap2 successfully addresses the issue of allelic bias, enabling more accurate downstream variant calls in repetitive sequences.

The complete sequence of a human genome.

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

A complete reference genome improves analysis of human genetic variation.

Compared to its predecessors, the Telomere-to-Telomere CHM13 genome adds nearly 200 million base pairs of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome for clinical and functional study. We show how this reference universally improves read mapping and variant calling for 3202 and 17 globally diverse samples sequenced with short and long reads, respectively. We identify hundreds of thousands of variants per sample in previously unresolved regions, showcasing the promise of the T2T-CHM13 reference for evolutionary and biomedical discovery. Simultaneously, this reference eliminates tens of thousands of spurious variants per sample, including reduction of false positives in 269 medically relevant genes by up to a factor of 12. Because of these improvements in variant discovery coupled with population and functional genomic resources, T2T-CHM13 is positioned to replace GRCh38 as the prevailing reference for human genetics.

KLF3 and PAX6 are candidate driver genes in late-stage, MSI-hypermutated endometrioid endometrial carcinomas.

Endometrioid endometrial carcinomas (EECs) are the most common histological subtype of uterine cancer. Late-stage disease is an adverse prognosticator for EEC. The purpose of this study was to analyze EEC exome mutation data to identify late-stage-specific statistically significantly mutated genes (SMGs), which represent candidate driver genes potentially associated with disease progression. We exome sequenced 15 late-stage (stage III or IV) non-ultramutated EECs and paired non-tumor DNAs; somatic variants were called using Strelka, Shimmer, SomaticSniper and MuTect. Additionally, somatic mutation calls were extracted from The Cancer Genome Atlas (TCGA) data for 66 late-stage and 270 early-stage (stage I or II) non-ultramutated EECs. MutSigCV (v1.4) was used to annotate SMGs in the two late-stage cohorts and to derive p-values for all mutated genes in the early-stage cohort. To test whether late-stage SMGs are statistically significantly mutated in early-stage tumors, q-values for late-stage SMGs were re-calculated from the MutSigCV (v1.4) early-stage p-values, adjusting for the number of late-stage SMGs tested. We identified 14 SMGs in the combined late-stage EEC cohorts. When the 14 late-stage SMGs were examined in the TCGA early-stage data, only Krüppel-like factor 3 (KLF3) and Paired box 6 (PAX6) failed to reach significance as early-stage SMGs, despite the inclusion of enough early-stage cases to ensure adequate statistical power. Within TCGA, nonsynonymous mutations in KLF3 and PAX6 were, respectively, exclusive or nearly exclusive to the microsatellite instability (MSI)-hypermutated molecular subgroup and were dominated by insertions-deletions at homopolymer tracts. In conclusion, our findings are hypothesis-generating and suggest that KLF3 and PAX6, which encode transcription factors, are MSI target genes and late-stage-specific SMGs in EEC.

Author Correction: A robust benchmark for detection of germline large deletions and insertions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Telomere-to-telomere assembly of a complete human X chromosome.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

Comparative clinical and genomic analysis of neurofibromatosis type 2-associated cranial and spinal meningiomas.

Neurofibromatosis type 2 (NF2) is an autosomal dominant Mendelian tumor predisposition disorder caused by germline pathogenic variants in the tumor suppressor NF2. Meningiomas are the second most common neoplasm in NF2, often occurring in multiple intracranial and spinal locations within the same patient. In this prospective longitudinal study, we assessed volumes and growth rates of ten spinal and ten cranial benign meningiomas in seven NF2 patients that concluded with surgical resection and performed whole-exome sequencing and copy-number variant (CNV) analysis of the tumors. Our comparison of the volume and the growth rate of NF2-associated spinal and cranial meningiomas point to the differences in timing of tumor initiation and/or to the differences in tumor progression (e.g., non-linear, saltatory growth) at these two anatomical locations. Genomic investigation of these tumors revealed that somatic inactivation of NF2 is the principal and perhaps the only driver of tumor initiation; and that tumor progression likely occurs via accumulation of CNVs, rather than point mutations. Results of this study contribute to a better understanding of NF2-associated meningiomas clinical behavior and their genetic underpinnings.

A robust benchmark for detection of germline large deletions and insertions.

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution and comprehensiveness. To help translate these methods to routine research and clinical practice, we developed a sequence-resolved benchmark set for identification of both false-negative and false-positive germline large insertions and deletions. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle Consortium integrated 19 sequence-resolved variant calling methods from diverse technologies. The final benchmark set contains 12,745 isolated, sequence-resolved insertion (7,281) and deletion (5,464) calls ≥50 base pairs (bp). The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.51 Gbp and 5,262 insertions and 4,095 deletions supported by ≥1 diploid assembly. We demonstrate that the benchmark set reliably identifies false negatives and false positives in high-quality SV callsets from short-, linked- and long-read sequencing and optical mapping.

Admixture mapping identifies genetic regions associated with blood pressure phenotypes in African Americans.

Hypertension occurs at a higher rate in African Americans than in European Americans. Based on the assumption that causal variants are more frequently found on DNA segments inherited from the ancestral population with higher disease risk, we employed admixture mapping to identify genetic loci with excess local African ancestry associated with blood pressure. Chromosomal regions 1q21.2-21.3, 4p15.1, 19q12 and 20p13 were significantly associated with diastolic blood pressure (ß = 5.28, -7.94, -6.82 and 5.89, P-value = 6.39E-04, 2.07E-04, 6.56E-05 and 5.04E-04, respectively); 1q21.2-21.3 and 19q12 were also significantly associated with mean arterial pressure (ß = 5.86 and -6.40, P-value = 5.32E-04 and 6.37E-04, respectively). We further selected SNPs that had large allele frequency differences within these regions and tested their association with blood pressure. SNP rs4815428 was significantly associated with diastolic blood pressure after Bonferroni correction (ß = -2.42, P-value = 9.57E-04), and it partially explained the admixture mapping signal at 20p13. SNPs rs771205 (ß = -1.99, P-value = 3.37E-03), rs3126067, rs2184953 and rs58001094 (the latter three exhibit strong linkage disequilibrium, ß = -2.3, P-value = 1.4E-03) were identified to be significantly associated with mean arterial pressure, and together they fully explained the admixture signal at 1q21.2-21.3. Although no SNP at 4p15.1 showed large ancestral allele frequency differences in our dataset, we detected association at low-frequency African-specific variants that mapped predominantly to the gene PCDH7, which is most highly expressed in aorta. Our results suggest that these regions may harbor genetic variants that contribute to the different prevalence of hypertension.

Low mutation burden and frequent loss of CDKN2A/B and SMARCA2, but not PRC2, define premalignant neurofibromatosis type 1-associated atypical neurofibromas.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a tumor-predisposition disorder caused by germline mutations in NF1. NF1 patients have an 8-16% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft-tissue sarcoma, often arising from preexisting benign plexiform neurofibromas (PNs) and atypical neurofibromas (ANFs). ANFs are distinct from both PN and MPNST, representing an intermediate step in malignant transformation. METHODS: In the first comprehensive genomic analysis of ANF originating from multiple patients, we performed tumor/normal whole-exome sequencing (WES) of 16 ANFs. In addition, we conducted WES of 3 MPNSTs, copy-number meta-analysis of 26 ANFs and 28 MPNSTs, and whole transcriptome sequencing analysis of 5 ANFs and 5 MPNSTs. RESULTS: We identified a low number of mutations (median 1, range 0-5) in the exomes of ANFs (only NF1 somatic mutations were recurrent), and frequent deletions of CDKN2A/B (69%) and SMARCA2 (42%). We determined that polycomb repressor complex 2 (PRC2) genes EED and SUZ12 were frequently mutated, deleted, or downregulated in MPNSTs but not in ANFs. Our pilot gene expression study revealed upregulated NRAS, MDM2, CCND1/2/3, and CDK4/6 in ANFs and MPNSTs, and overexpression of EZH2 in MPNSTs only. CONCLUSIONS: The PN-ANF transition is primarily driven by the deletion of CDKN2A/B. Further progression from ANF to MPNST likely involves broad chromosomal rearrangements and frequent inactivation of the PRC2 genes, loss of the DNA repair genes, and copy-number increase of signal transduction and cell-cycle and pluripotency self-renewal genes.

The Sleep Inbred Panel, a Collection of Inbred Drosophila melanogaster with Extreme Long and Short Sleep Duration.

Understanding how genomic variation causes differences in observable phenotypes remains a major challenge in biology. It is difficult to trace the sequence of events originating from genomic variants to changes in transcriptional responses or protein modifications. Ideally, one would conduct experiments with individuals that are at either extreme of the trait of interest, but such resources are often not available. Further, advances in genome editing will enable testing of candidate polymorphisms individually and in combination. Here we have created a resource for the study of sleep with 39 inbred lines of Drosophila-the Sleep Inbred Panel (SIP). SIP lines have stable long- and short-sleeping phenotypes developed from naturally occurring polymorphisms. These lines are fully sequenced, enabling more accurate targeting for genome editing and transgenic constructs. This panel facilitates the study of intermediate transcriptional and proteomic correlates of sleep, and supports genome editing studies to verify polymorphisms associated with sleep duration.

NovoGraph: Human genome graph construction from multiple long-read de novo assemblies.

Genome graphs are emerging as an important novel approach to the analysis of high-throughput human sequencing data. By explicitly representing genetic variants and alternative haplotypes in a mappable data structure, they can enable the improved analysis of structurally variable and hyperpolymorphic regions of the genome. In most existing approaches, graphs are constructed from variant call sets derived from short-read sequencing. As long-read sequencing becomes more cost-effective and enables de novo assembly for increasing numbers of whole genomes, a method for the direct construction of a genome graph from sets of assembled human genomes would be desirable. Such assembly-based genome graphs would encompass the wide spectrum of genetic variation accessible to long-read-based de novo assembly, including large structural variants and divergent haplotypes. Here we present NovoGraph, a method for the construction of a human genome graph directly from a set of de novo assemblies. NovoGraph constructs a genome-wide multiple sequence alignment of all input contigs and creates a graph by merging the input sequences at positions that are both homologous and sequence-identical. NovoGraph outputs resulting graphs in VCF format that can be loaded into third-party genome graph toolkits. To demonstrate NovoGraph, we construct a genome graph with 23,478,835 variant sites and 30,582,795 variant alleles from de novo assemblies of seven ethnically diverse human genomes (AK1, CHM1, CHM13, HG003, HG004, HX1, NA19240). Initial evaluations show that mapping against the constructed graph reduces the average mismatch rate of reads from sample NA12878 by approximately 0.2%, albeit at a slightly increased rate of reads that remain unmapped.

The FOXA2 transcription factor is frequently somatically mutated in uterine carcinosarcomas and carcinomas.

BACKGROUND: Uterine carcinosarcomas (UCSs) are a rare but clinically aggressive form of cancer. They are biphasic tumors consisting of both epithelial and sarcomatous components. The majority of uterine carcinosarcomas are clonal, with the carcinomatous cells undergoing metaplasia to give rise to the sarcomatous component. The objective of the current study was to identify novel somatically mutated genes in UCSs. METHODS: We whole exome sequenced paired tumor and nontumor DNAs from 14 UCSs and orthogonally validated 464 somatic variants using Sanger sequencing. Fifteen genes that were somatically mutated in at least 2 tumor exomes were Sanger sequenced in another 39 primary UCSs. RESULTS: Overall, among 53 UCSs in the current study, the most frequently mutated of these 15 genes were tumor protein p53 (TP53) (75.5%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (34.0%), protein phosphatase 2, regulatory subunit A, alpha (PPP2R1A) (18.9%), F-box and WD repeat domain containing 7 (FBXW7) (18.9%), chromodomain helicase DNA binding protein 4 (CHD4) (17.0%), and forkhead box A2 (FOXA2) (15.1%). FOXA2 has not previously been implicated in UCSs and was predominated by frameshift and nonsense mutations. One UCS with a FOXA2 frameshift mutation expressed truncated FOXA2 protein by immunoblotting. Sequencing of FOXA2 in 160 primary endometrial carcinomas revealed somatic mutations in 5.7% of serous, 22.7% of clear cell, 9% of endometrioid, and 11.1% of mixed endometrial carcinomas, the majority of which were frameshift mutations. CONCLUSIONS: Collectively, the findings of the current study provide compelling genetic evidence that FOXA2 is a pathogenic driver gene in the etiology of primary uterine cancers, including UCSs. Cancer 2018;124:65-73. © 2017 American Cancer Society.

Selection for long and short sleep duration in Drosophila melanogaster reveals the complex genetic network underlying natural variation in sleep.

Why do some individuals need more sleep than others? Forward mutagenesis screens in flies using engineered mutations have established a clear genetic component to sleep duration, revealing mutants that convey very long or short sleep. Whether such extreme long or short sleep could exist in natural populations was unknown. We applied artificial selection for high and low night sleep duration to an outbred population of Drosophila melanogaster for 13 generations. At the end of the selection procedure, night sleep duration diverged by 9.97 hours in the long and short sleeper populations, and 24-hour sleep was reduced to 3.3 hours in the short sleepers. Neither long nor short sleeper lifespan differed appreciably from controls, suggesting little physiological consequences to being an extreme long or short sleeper. Whole genome sequence data from seven generations of selection revealed several hundred thousand changes in allele frequencies at polymorphic loci across the genome. Combining the data from long and short sleeper populations across generations in a logistic regression implicated 126 polymorphisms in 80 candidate genes, and we confirmed three of these genes and a larger genomic region with mutant and chromosomal deficiency tests, respectively. Many of these genes could be connected in a single network based on previously known physical and genetic interactions. Candidate genes have known roles in several classic, highly conserved developmental and signaling pathways-EGFR, Wnt, Hippo, and MAPK. The involvement of highly pleiotropic pathway genes suggests that sleep duration in natural populations can be influenced by a wide variety of biological processes, which may be why the purpose of sleep has been so elusive.

Somatic mutation profiles of clear cell endometrial tumors revealed by whole exome and targeted gene sequencing.

BACKGROUND: The molecular pathogenesis of clear cell endometrial cancer (CCEC), a tumor type with a relatively unfavorable prognosis, is not well defined. We searched exome-wide for novel somatically mutated genes in CCEC and assessed the mutational spectrum of known and candidate driver genes in a large cohort of cases. METHODS: We conducted whole exome sequencing of paired tumor-normal DNAs from 16 cases of CCEC (12 CCECs and the CCEC components of 4 mixed histology tumors). Twenty-two genes-of-interest were Sanger-sequenced from another 47 cases of CCEC. Microsatellite instability (MSI) and microsatellite stability (MSS) were determined by genotyping 5 mononucleotide repeats. RESULTS: Two tumor exomes had relatively high mutational loads and MSI. The other 14 tumor exomes were MSS and had 236 validated nonsynonymous or splice junction somatic mutations among 222 protein-encoding genes. Among the 63 cases of CCEC in this study, we identified frequent somatic mutations in TP53 (39.7%), PIK3CA (23.8%), PIK3R1 (15.9%), ARID1A (15.9%), PPP2R1A (15.9%), SPOP (14.3%), and TAF1 (9.5%), as well as MSI (11.3%). Five of 8 mutations in TAF1, a gene with no known role in CCEC, localized to the putative histone acetyltransferase domain and included 2 recurrently mutated residues. Based on patterns of MSI and mutations in 7 genes, CCEC subsets molecularly resembled serous endometrial cancer (SEC) or endometrioid endometrial cancer (EEC). CONCLUSION: Our findings demonstrate molecular similarities between CCEC and SEC and EEC and implicate TAF1 as a novel candidate CCEC driver gene. Cancer 2017;123:3261-8. © 2017 American Cancer Society.

First insight into the somatic mutation burden of neurofibromatosis type 2-associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation.

BACKGROUND: Neurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2-associated meningiomas remains largely unexamined. CASE PRESENTATION: Here, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copy-number analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivation of NF2, confirming the central role of the gene in meningioma formation. The genome of the benign tumor closely resembled that of a normal diploid cell and had only one other deleterious mutation (EPHB3). In contrast, the chromosomal architecture of the grade II tumor was highly re-arranged, yet uniform among all analyzed fragments, implying that this large and fast growing tumor was composed of relatively few clones. Besides multiple gains and losses, the grade II meningioma harbored numerous chromosomal translocations. WES analysis of the atypical tumor identified deleterious mutations in two genes: ADAMTSL3 and CAPN5 in all fragments, indicating that the mutations were present in the cell undergoing fast clonal expansion CONCLUSIONS: This is the first WES study of NF2-associated meningiomas. Besides second NF2 copy inactivation, we found low somatic burden in both tumors and high level of genomic instability in the atypical meningioma. Genomic instability resulting in altered gene dosage and compromised structural integrity of multiple genes may be the primary reason of the high growth rate for the grade II tumor. Further study of ADAMTSL3 and CAPN5 may lead to elucidation of their molecular implications in meningioma pathogenesis.

iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations.

Genome integrity of induced pluripotent stem cells (iPSCs) has been extensively studied in recent years, but it is still unclear whether iPSCs contain more genomic variations than cultured somatic cells. One important question is the origin of genomic variations detected in iPSCs-whether iPSC reprogramming induces such variations. Here, we undertook a unique approach by deriving fibroblast subclones and clonal iPSC lines from the same fibroblast population and applied next-generation sequencing to compare genomic variations in these lines. Targeted deep sequencing of parental fibroblasts revealed that most variants detected in clonal iPSCs and fibroblast subclones were rare variants inherited from the parental fibroblasts. Only a small number of variants remained undetectable in the parental fibroblasts, which were thus likely to be de novo. Importantly, the clonal iPSCs and fibroblast subclones contained comparable numbers of de novo variants. Collectively, our data suggest that iPSC reprogramming is not mutagenic.

Variant Calling From Next Generation Sequence Data.

The use of next generation nucleotide sequencing to discover and genotype small sequence variants has led to numerous insights into the molecular causes of various diseases. This chapter describes the use of freely available software to align next generation sequencing reads to a reference and then to use the resulting alignments to call, annotate, view, and filter small sequence variants. The suggested variant calling workflow includes read alignment with novoalign, the removal of polymerase chain reaction duplicate sequences with samtools or bamUtils, and the detection of variants with Freebayes or bam2mpg software. ANNOVAR is then used to annotate the predicted variants using gene models, population frequencies, and predicted mutation severity, producing variant files which can be viewed and filtered with the variant display tool VarSifter.