Benchmarking challenging small variants with linked and long reads.

Genome in a Bottle benchmarks are widely used to help validate clinical sequencing pipelines and develop variant calling and sequencing methods. Here we use accurate linked and long reads to expand benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are challenging for short reads. These benchmarks add more than 300,000 SNVs and 50,000 insertions or deletions (indels) and include 16% more exonic variants, many in challenging, clinically relevant genes not covered previously, such as PMS2. For HG002, we include 92% of the autosomal GRCh38 assembly while excluding regions problematic for benchmarking small variants, such as copy number variants, that should not have been in the previous version, which included 85% of GRCh38. It identifies eight times more false negatives in a short read variant call set relative to our previous benchmark. We demonstrate that this benchmark reliably identifies false positives and false negatives across technologies, enabling ongoing methods development.

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.

Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology.

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the linker sequence for the mature LASSO probe through Cre-LoxP intramolecular recombination. This strategy generates high quality LASSO probes libraries (≈46% of correct probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 Escherichia coli ORFs spanning from 400- to 5000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, were able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

Shotgun transcriptome, spatial omics, and isothermal profiling of SARS-CoV-2 infection reveals unique host responses, viral diversification, and drug interactions.

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin-angiotensin-aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

Genomic and immunologic correlates of LAG-3 expression in cancer.

Immune checkpoint blockade leads to unprecedented responses in many cancers. Although currently available agents mostly target the PD-1 and CTLA-4 pathways, agents targeting the immune checkpoint protein LAG-3 are under active clinical development, and early clinical data show that LAG-3 expression is a biomarker of response to LAG-3 blockade. To determine which cancers may benefit most from LAG-3 blockade, we performed a pan-cancer analysis of The Cancer Genome Atlas dataset to identify genomic and immunologic correlates of LAG-3 expression. High mutation burden, and expression of exogenous virus (EBV, HPV) or endogenous retrovirus (ERV3-2), were associated with overexpression of LAG-3 in multiple cancers. Although CD8+ T-cell marker (CD8A) and LAG-3 were strongly co-expressed with each other and with PD-L1 in most cancers, there were three notable exceptions: HPV+ head-neck squamous cell cancer, renal cell cancer, and glioblastoma. These results may have important implications for guiding development clinical trials of LAG-3 blockade.

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.

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.

Single-cell sperm transcriptomes and variants from fathers of children with and without autism spectrum disorder.

The human sperm is one of the smallest cells in the body, but also one of the most important, as it serves as the entire paternal genetic contribution to a child. Investigating RNA and mutations in sperm is especially relevant for diseases such as autism spectrum disorders (ASD), which have been correlated with advanced paternal age. Historically, studies have focused on the assessment of bulk sperm, wherein millions of individual sperm are present and only high-frequency variants can be detected. Using 10× Chromium single-cell sequencing technology, we assessed the transcriptome from >65,000 single spermatozoa across six sperm donors (scSperm-RNA-seq), including two who fathered multiple children with ASD and four fathers of neurotypical children. Using RNA-seq methods for differential expression and variant analysis, we found clusters of sperm mutations in each donor that are indicative of the sperm being produced by different stem cell pools. Finally, we have shown that genetic variations can be found in single sperm.

IGF2 drives formation of ileal neuroendocrine tumors in patients and mice.

By the strictest of definitions, a genetic driver of tumorigenesis should fulfill two criteria: it should be altered in a high percentage of patient tumors, and it should also be able to cause the same type of tumor to form in mice. No gene that fits either of these criteria has ever been found for ileal neuroendocrine tumors (I-NETs), which in humans are known for an unusual lack of recurrently mutated genes, and which have never been detected in mice. In the following report, we show that I-NETs can be generated by transgenic RT2 mice, which is a classic model for a genetically unrelated disease, pancreatic neuroendocrine tumors (PNETs). The ability of RT2 mice to generate I-NETs depended upon genetic background. I-NETs appeared in a B6AF1 genetic background, but not in a B6 background nor even in an AB6F1 background. AB6F1 and B6AF1 have identical nuclear DNA but can potentially express different allelic forms of imprinted genes. This led us to test human I-NETs for loss of imprinting, and we discovered that the IGF2 gene showed loss of imprinting and increased expression in the I-NETs of 57% of patients. By increasing IGF2 activity genetically, I-NETs could be produced by RT2 mice in a B6 genetic background, which otherwise never developed I-NETs. The facts that IGF2 is altered in a high percentage of patients with I-NETs and that I-NETs can form in mice that have elevated IGF2 activity, define IGF2 as the first genetic driver of ileal neuroendocrine tumorigenesis.

Whole Genome Sequencing and Assembly of the Asian Honey Bee Apis dorsata.

The Asian honey bee (Apis dorsata) is distinct from its more widely distributed cousin Apis mellifera by a few key characteristics. Most prominently, A. dorsata, nest in the open by forming a colony clustered around the honeycomb, whereas A. mellifera nest in concealed cavities. Additionally, the worker and reproductive castes are all of the same size in A. dorsata. In order to investigate these differences, we performed whole genome sequencing of A. dorsata using a hybrid Oxford Nanopore and Illumina approach. The 223 Mb genome has an N50 of 35 kb with the largest scaffold of 302 kb. We have found that there are many genes in the dorsata genome that are distinct from other hymenoptera and also large amounts of transposable elements, and we suggest some candidate genes for A. dorsata's exceptional level of defensive aggression.

TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer.

In treating patients with castration resistant prostate cancer (CRPC), enzalutamide, the second-generation androgen receptor (AR) antagonist, is an accepted standard of care. However, clinical benefits are limited to a median time of 4.8 months because resistance inevitably emerges. To determine the mechanism of treatment resistance, we carried out a RNA sequence analysis and found increased expression levels of neuroendocrine markers in the enzalutamide-resistant LNCaP human prostate cancer (CaP) cell line when compared to the parental cell line. Subsequent studies demonstrated that Transcription Factor-4 (TCF4), a transcription factor implicated in WNT signaling, mediated neuroendocrine differentiation (NED) in response to enzalutamide treatment and was elevated in the enzalutamide-resistant LNCaP. In addition, we observed that PTHrP mediated enzalutamide resistance in tissue culture and inducible TCF4 overexpression resulted in enzalutamide-resistance in a mouse xenograft model. Finally, small molecule inhibitors of TCF4 or PTHrP partially reversed enzalutamide resistance in CaP cells. When tissues obtained from men who died of metastatic CaP were examined, a positive correlation was found between the expression levels of TCF4 and PTHrP. Taken together, the current results indicate that TCF4 induces enzalutamide resistance via NED in CaP.

Genome content analysis yields new insights into the relationship between the human malaria parasite Plasmodium falciparum and its anopheline vectors.

BACKGROUND: The persistent and growing gap between the availability of sequenced genomes and the ability to assign functions to sequenced genes led us to explore ways to maximize the information content of automated annotation for studies of anopheline mosquitos. Specifically, we use genome content analysis of a large number of previously sequenced anopheline mosquitos to follow the loss and gain of protein families over the evolutionary history of this group. The importance of this endeavor lies in the potential for comparative genomic studies between Anopheles and closely related non-vector species to reveal ancestral genome content dynamics involved in vector competence. In addition, comparisons within Anopheles could identify genome content changes responsible for variation in the vectorial capacity of this family of important parasite vectors. RESULTS: The competence and capacity of P. falciparum vectors do not appear to be phylogenetically constrained within the Anophelinae. Instead, using ancestral reconstruction methods, we suggest that a previously unexamined component of vector biology, anopheline nucleotide metabolism, may contribute to the unique status of anophelines as P. falciparum vectors. While the fitness effects of nucleotide co-option by P. falciparum parasites on their anopheline hosts are not yet known, our results suggest that anopheline genome content may be responding to selection pressure from P. falciparum. Whether this response is defensive, in an attempt to redress improper nucleotide balance resulting from P. falciparum infection, or perhaps symbiotic, resulting from an as-yet-unknown mutualism between anophelines and P. falciparum, is an open question that deserves further study. CONCLUSIONS: Clearly, there is a wealth of functional information to be gained from detailed manual genome annotation, yet the rapid increase in the number of available sequences means that most researchers will not have the time or resources to manually annotate all the sequence data they generate. We believe that efforts to maximize the amount of information obtained from automated annotation can help address the functional annotation deficit that most evolutionary biologists now face, and here demonstrate the value of such an approach.

A whole genome gene content phylogenetic analysis of anopheline mosquitoes.

Construction of stringent gene content matrices was accomplished for 21 Anopheline mosquito species and strains and four outgroups species. The presence absence matrix using e-75 as a cutoff in single linkage clustering had over 17,000 ortholog groups. We used the gene content matrix to generate a phylogenetic hypothesis that is in general agreement with gene sequence based phylogenies. In addition to establishing a congruent gene content phylogeny we examined the consistency of three methods for analyzing presence absence data - unweighted parsimony, dollo parsimonly and maximum likelihood using a BINGAMMA model. An examination of the chromosomal location of the gains and losses in the presence absence matrix revealed a low frequency of gains and losses at centromeres and tips of chromosomes.

The mitogenome of the bed bug Cimex lectularius (Hemiptera: Cimicidae).

We report the extraction of a bed bug mitogenome from high-throughput sequencing projects originally focused on the nuclear genome of Cimex lectularius. The assembled mitogenome has a similar AT nucleotide composition bias found in other insects. Phylogenetic analysis of all protein-coding genes indicates that C. lectularius is clearly a member of a paraphyletic Cimicomorpha clade within the Order Hemiptera.

Genome assembly and geospatial phylogenomics of the bed bug Cimex lectularius.

The common bed bug (Cimex lectularius) has been a persistent pest of humans for thousands of years, yet the genetic basis of the bed bug's basic biology and adaptation to dense human environments is largely unknown. Here we report the assembly, annotation and phylogenetic mapping of the 697.9-Mb Cimex lectularius genome, with an N50 of 971 kb, using both long and short read technologies. A RNA-seq time course across all five developmental stages and male and female adults generated 36,985 coding and noncoding gene models. The most pronounced change in gene expression during the life cycle occurs after feeding on human blood and included genes from the Wolbachia endosymbiont, which shows a simultaneous and coordinated host/commensal response to haematophagous activity. These data provide a rich genetic resource for mapping activity and density of C. lectularius across human hosts and cities, which can help track, manage and control bed bug infestations.

Insect genome content phylogeny and functional annotation of core insect genomes.

Twenty-one fully sequenced and well annotated insect genomes were examined for genome content in a phylogenetic context. Gene presence/absence matrices and phylogenetic trees were constructed using several phylogenetic criteria. The role of e-value on phylogenetic analysis and genome content characterization is examined using scaled e-value cutoffs and a single linkage clustering approach to orthology determination. Previous studies have focused on the role of gene loss in terminals in the insect tree of life. The present study examines several common ancestral nodes in the insect tree. We suggest that the common ancestors of major insect groups like Diptera, Hymenoptera, Hemiptera and Holometabola experience more gene gain than gene loss. This suggests that as major insect groups arose, their genomic repertoire expanded through gene duplication (segmental duplications), followed by contraction by gene loss in specific terminal lineages. In addition, we examine the functional significance of the loss and gain of genes in the divergence of some of the major insect groups.

The impact of read length on quantification of differentially expressed genes and splice junction detection.

BACKGROUND: The initial next-generation sequencing technologies produced reads of 25 or 36 bp, and only from a single-end of the library sequence. Currently, it is possible to reliably produce 300 bp paired-end sequences for RNA expression analysis. While read lengths have consistently increased, people have assumed that longer reads are more informative and that paired-end reads produce better results than single-end reads. We used paired-end 101 bp reads and trimmed them to simulate different read lengths, and also separated the pairs to produce single-end reads. For each read length and paired status, we evaluated differential expression levels between two standard samples and compared the results to those obtained by qPCR. RESULTS: We found that, with the exception of 25 bp reads, there is little difference for the detection of differential expression regardless of the read length. Once single-end reads are at a length of 50 bp, the results do not change substantially for any level up to, and including, 100 bp paired-end. However, splice junction detection significantly improves as the read length increases with 100 bp paired-end showing the best performance. We performed the same analysis on two ENCODE samples and found consistent results confirming that our conclusions have broad application. CONCLUSIONS: A researcher could save substantial resources by using 50 bp single-end reads for differential expression analysis instead of using longer reads. However, splicing detection is unquestionably improved by paired-end and longer reads. Therefore, an appropriate read length should be used based on the final goal of the study.

Integrative annotation of variants from 1092 humans: application to cancer genomics.

Interpreting variants, especially noncoding ones, in the increasing number of personal genomes is challenging. We used patterns of polymorphisms in functionally annotated regions in 1092 humans to identify deleterious variants; then we experimentally validated candidates. We analyzed both coding and noncoding regions, with the former corroborating the latter. We found regions particularly sensitive to mutations ("ultrasensitive") and variants that are disruptive because of mechanistic effects on transcription-factor binding (that is, "motif-breakers"). We also found variants in regions with higher network centrality tend to be deleterious. Insertions and deletions followed a similar pattern to single-nucleotide variants, with some notable exceptions (e.g., certain deletions and enhancers). On the basis of these patterns, we developed a computational tool (FunSeq), whose application to ~90 cancer genomes reveals nearly a hundred candidate noncoding drivers.

Pervasive sequence patents cover the entire human genome.

The scope and eligibility of patents for genetic sequences have been debated for decades, but a critical case regarding gene patents (Association of Molecular Pathologists v. Myriad Genetics) is now reaching the US Supreme Court. Recent court rulings have supported the assertion that such patents can provide intellectual property rights on sequences as small as 15 nucleotides (15mers), but an analysis of all current US patent claims and the human genome presented here shows that 15mer sequences from all human genes match at least one other gene. The average gene matches 364 other genes as 15mers; the breast-cancer-associated gene BRCA1 has 15mers matching at least 689 other genes. Longer sequences (1,000 bp) still showed extensive cross-gene matches. Furthermore, 15mer-length claims from bovine and other animal patents could also claim as much as 84% of the genes in the human genome. In addition, when we expanded our analysis to full-length patent claims on DNA from all US patents to date, we found that 41% of the genes in the human genome have been claimed. Thus, current patents for both short and long nucleotide sequences are extraordinarily non-specific and create an uncertain, problematic liability for genomic medicine, especially in regard to targeted re-sequencing and other sequence diagnostic assays.

Implication of a rare deletion at distal 16p11.2 in schizophrenia.

CONTEXT: Large genomic copy number variations have been implicated as strong risk factors for schizophrenia. However, the rarity of these events has created challenges for the identification of further pathogenic loci, and extremely large samples are required to provide convincing replication. OBJECTIVE: To detect novel copy number variations that increase the susceptibility to schizophrenia by using 2 ethnically homogeneous discovery cohorts and replication in large samples. DESIGN: Genetic association study of microarray data. SETTING: Samples of DNA were collected at 9 sites from different countries. PARTICIPANTS: Two discovery cohorts consisted of 790 cases with schizophrenia and schizoaffective disorder and 1347 controls of Ashkenazi Jewish descent and 662 parent-offspring trios from Bulgaria, of which the offspring had schizophrenia or schizoaffective disorder. Replication data sets consisted of 12,398 cases and 17,945 controls. MAIN OUTCOME MEASURES: Statistically increased rate of specific copy number variations in cases vs controls. RESULTS: One novel locus was implicated: a deletion at distal 16p11.2, which does not overlap the proximal 16p11.2 locus previously reported in schizophrenia and autism. Deletions at this locus were found in 13 of 13,850 cases (0.094%) and 3 of 19,954 controls (0.015%) (odds ratio, 6.25 [95% CI, 1.78-21.93]; P = .001, Fisher exact test). CONCLUSIONS: Deletions at distal 16p11.2 have been previously implicated in developmental delay and obesity. The region contains 9 genes, several of which are implicated in neurological diseases, regulation of body weight, and glucose homeostasis. A telomeric extension of the deletion, observed in about half the cases but no controls, potentially implicates an additional 8 genes. Our findings add a new locus to the list of copy number variations that increase the risk for development of schizophrenia.