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Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line–derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology., Editorial summary: Reliable detection of mutations below 0.5% variant allele frequency remains a key challenge for circulating tumor DNA sequencing assays.

Clinical oncology heavily relies on formalin-fixed, paraffin-embedded (FFPE) tissue samples for histology and molecular characterization. The chemical and physical modifications of nucleic acids introduced during fixation, storage and purification negatively impact molecular profiling and vary from sample-to-sample. Conventional sonication and ligation-based library preparation is considered the gold-standard approach for FFPE samples, but it is time-consuming and expensive. Importantly, these processes introduce artifacts that impact downstream analysis and interpretation (Haile et al., 2019). We have developed a novel fragmentation chemistry that virtually eliminates hairpin artifacts, achieving levels on par with non-FFPE control samples. Our fragmentation method is highly scalable, exhibits minimal sequence bias, and reduces cost and workflow inefficiencies associated with mechanical shearing. In this study, we developed a unified method for library preparation from FFPE samples which produces similar insert sizes across variable input mass and quality of FFPE samples. We carefully evaluated the performance of this method relative to a sonication-based approach employing the KAPA HyperPrep kit. Libraries were constructed from 50 to 200 ng of FFPE DNA, inputs typically used for NGS, ranging from low-to high-quality as assessed using a qPCR-based method and DNA integrity (DIN). Targeted sequencing was performed using a 37 kb custom oncology hybridization capture panel to investigate molecular complexity. Our workflow virtually eliminated hairpin artifacts that were present in up to 4.5% of reads in sonication-based libraries. Soft-clipping was also 3- to 7-fold lower in libraries prepared with the Watchmaker kit relative to sonicated DNA libraries, improving overall sequencing economy. Furthermore, the mean target coverage achieved with the Watchmaker kit was comparable to or higher than sonication libraries using the same input mass. Because input masses were normalized post-shearing, which typically results in 20-40% sample loss, coverage with our approach is significantly higher relative to sonication, if normalizing to pre-sheared DNA input. Watchmaker DNA Library Preparation with Fragmentation enables high-quality DNA library preparation from damaged FFPE samples, producing high target coverage, uniform insert size, and minimizing sequencing artifacts to improve sensitivity and specificity. This approach is highly scalable and automatable, enabling various oncology applications. Citation Format: Giulia Corbet, Philip Benson, Kailee Reed, Skyler Mishkin, Thomas Harrison, Kristin Scott, Zane Jaafar, Kristina Giorda, Josh Haimes, Martin Ranik, Brian Kudlow. A novel enzymatic library preparation workflow that dramatically reduces artifacts associated with damaged FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 215.

Background The olive fruit fly, Bactrocera oleae, is the most important pest in the olive fruit agribusiness industry. This is because female flies lay their eggs in the unripe fruits and upon hatching the larvae feed on the fruits thus destroying them. The lack of a high-quality genome and other genomic and transcriptomic data has hindered progress in understanding the fly’s biology and proposing alternative control methods to pesticide use. Results Genomic DNA was sequenced from male and female Demokritos strain flies, maintained in the laboratory for over 45 years. We used short-, mate-pair-, and long-read sequencing technologies to generate a combined male-female genome assembly (GenBank accession GCA_001188975.2). Genomic DNA sequencing from male insects using 10x Genomics linked-reads technology followed by mate-pair and long-read scaffolding and gap-closing generated a highly contiguous 489 Mb genome with a scaffold N50 of 4.69 Mb and L50 of 30 scaffolds (GenBank accession GCA_001188975.4). RNA-seq data generated from 12 tissues and/or developmental stages allowed for genome annotation. Short reads from both males and females and the chromosome quotient method enabled identification of Y-chromosome scaffolds which were extensively validated by PCR. Conclusions The high-quality genome generated represents a critical tool in olive fruit fly research. We provide an extensive RNA-seq data set, and genome annotation, critical towards gaining an insight into the biology of the olive fruit fly. In addition, elucidation of Y-chromosome sequences will advance our understanding of the Y-chromosome’s organization, function and evolution and is poised to provide avenues for sterile insect technique approaches.

Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

Despite tremendous progress in genome sequencing, the basic goal of producing a phased (haplotype-resolved) genome sequence with end-to-end contiguity for each chromosome at reasonable cost and effort is still unrealized. In this study, we describe an approach to performing de novo genome assembly and experimental phasing by integrating the data from Illumina short-read sequencing, 10X Genomics linked-read sequencing, and BioNano Genomics genome mapping to yield a high-quality, phased, de novo assembled human genome.

Rare gene knockouts in adult humans On average, most people's genomes contain approximately 100 completely nonfunctional genes. These loss-of-function (LOF) mutations tend to be rare and/or occur only as a single copy within individuals. Narasimhan et al. investigated LOF in a Pakistani population with high levels of consanguinity. Examining LOF alleles that were identical by descent, they found, as expected, an absence of homozygote LOF for certain protein-coding genes. However, they also identified many homozygote LOF alleles with no apparent deleterious phenotype, including some that were expected to confer genetic disease. Indeed, one family had lost the recombination-associated gene PRDM9 . Science , this issue p. 474

Consisting of only ~2% of the human genome, the exome accounts for ~85% of genetic disorders. Efficient sequencing of the human exome with complete and high coverage depth at low cost is invaluable for furthering research in clinical applications. IDT's xGen Exome Panel has proven to be a high performing option. Here, we present the updated xGen Exome Research Panel v2.0 in direct comparison with two other leading commercial human exome panels, using workflows per manufacturer's specifications. NA12878 genomic DNA libraries were pooled together for 8-plex captures for all three platforms and sequenced on the Illumina NextSeq 500. Equivalent number of reads per sample were analyzed against a universal human exome target space to compare across the different exome panels. IDT's Exome NGS solution provided significantly highest on-target percentage at >90% as well as the greatest depth of coverage at >96% bases covered at >20X and >98% bases covered at >10X. Importantly, IDT's platform also reported the most complete gene-level coverage, demonstrated by minimal exon drop-outs in difficult-to-target genes. While 8-plex is the upper limit supported by other suppliers, IDT's platform supports 12-plex workflow. The higher multiplex in combination with high coverage and on-target performance enables IDT to present the lowest total sequencing cost per sample. Since IDT hybridization capture baits are individually synthesized and qualified with the same high standards as standalone oligonucleotide products, lot-to-lot variability is negligible. This presents researchers with an option they can rely on for long-term use and places the focus on the true variabilities of the sample. In conclusion, this study demonstrates xGen Exome Research Panel v2.0, when combined with IDT's DNA Library Prep Kit, provides researchers with a complete Exome NGS solution that is competitive both in performance and sequencing cost. Citation Format: Manqing Hong, Bosun Min, Nicole Roseman, Ekaterina Star, Timothy Rusch, Krishnalekha Datta, Steve Groenewold, Longhui Ren, Jinglie Zhou, Kevin Lai, Xiaohui Wang, Nick Downey, Kristina Giorda, Alexandra Wang, Yu Wang, Lynette A. Lewis, Patrick J. Lau, Steven Henck. Improved human exome sequencing workflow with the most complete coverage [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1349.

Long-read sequencing has greatly contributed to the generation of high quality assemblies, albeit at a high cost. It is also not always clear how to combine sequencing platforms. We sequenced the genome of the olive fruit fly (Bactrocera oleae), the most important pest in the olive fruits agribusiness industry, using Illumina short-reads, mate-pairs, 10x Genomics linked-reads, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT). The 10x linked-reads assembly gave the most contiguous assembly with an N50 of 2.16 Mb. Scaffolding the linked-reads assembly using long-reads from ONT gave a more contiguous assembly with scaffold N50 of 4.59 Mb. We also present the most extensive transcriptome datasets of the olive fly derived from different tissues and stages of development. Finally, we used the Chromosome Quotient method to identify Y-chromosome scaffolds and show that the long-reads based assembly generates very highly contiguous Y-chromosome assembly.JR is a member of the MinION Access Program (MAP) and has received free-of-charge flow cells and sequencing kits from Oxford Nanopore Technologies for other projects. JR has had no other financial support from ONT.AB has received re-imbursement for travel costs associated with attending Nanopore Community meeting 2018, a meeting organized my Oxford Nanopore Technologies.

Background Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. Results Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. Conclusions For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications. Electronic supplementary material The online version of this article (10.1186/s12864-017-4428-5) contains supplementary material, which is available to authorized users.

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.

Large-scale cancer profiling using next generation sequencing (NGS) has become instrumental to the discovery and identification of new, targetable cancer alterations. A comprehensive set of 532 oncogene targets were combined to create the new xGen® Pan-Cancer Panel V2 for hybrid capture sequencing. This xGen panel covers 2.2 Mb of the human genome, and allows for the simultaneous detection of copy number variations (CNVs), insertions and deletions (indels), gene rearrangements, and microsatellite instability across a wide range of sample types, inputs, and quality. Using a new library prep workflow optimized for low quality samples and low input, panel performance was first evaluated with 30 ng of input DNA using libraries built from matched samples [formalin-fixed paraffin-embedded (FFPE) tumor gDNA, frozen adjacent normal tissue gDNA, and cell-free DNA (cfDNA)] from five lung cancer donors (n = 15). Sample quality ranged from a mean DIN of 4.4 ±1.1 to 8.3 ±0.9 for FFPE tumor gDNA and frozen normal gDNA, respectively. After subsampling to 200X mean target coverage, 96% of target bases had at least 40X coverage for all libraries. Comparative hierarchal clustering analysis was then used to identify lung cancer mutations shared in all tumor samples. NGS gDNA reference standards from Horizon Discovery (HD753, HD798, HD799, and HD803) with verified CNVs, single nucleotide variations (SNVs), amplifications, and fusions, and were used to evaluate detection rates at different library input masses down to 1 ng. cDNA libraries were also prepared from RNA extracted from FFPE 5-Fusion RNA Multiplex Reference Standards (HD796, HD783). We identified all possible gene fusion events in the positive control using the structural variant caller, LUMPY (https://github.com/arq5x/lumpy-sv). The xGen Pan-Cancer Panel V2 enables a cost-efficient and time-saving approach for the detection of multiple oncogene targets. Citation Format: Katharine Dilger, Yongming Sun, Kevin Lai, Ushati Das Chakravarty, Nicole Sponer, Kristina Giorda, Patrick Lau, Yu Wang. DNA-based fusion detection using a pan-cancer tumor profiling 532-oncogene panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4348.

Diagnostic tools based on next generation sequencing are fundamentally transforming clinical oncology. However, there is a lack of adequate library preparation strategies for highly degraded, clinically relevant samples, such as cell-free DNA (cfDNA) and formalin-fixed paraffin-embedded (FFPE) DNA. Due to the extreme heterogeneity of these sample types, targeted sequencing is often used to achieve deep coverage of genomic loci and enable detection of low-frequency variants. Commercially available protocols for library preparation require stringent size-selection to remove adapter-dimers, which reduces library complexity and variant detection power. Achieving high specificity can be challenging because low-frequency artifacts arise from a variety of sources, including DNA extraction, library construction, PCR, hybrid selection, and sequencing. These artifacts can be identified by “duplex sequencing”, where strand-specific unique molecular identifiers (UMIs) are used to confirm the presence of an alteration on both strands of an input molecule. However, duplex sequencing typically delivers low conversion rates with degraded samples due to poor ligation efficiency and template loss during size-selection. Here, we present the IDT library preparation kit optimized for low-input and degraded samples. Our novel library construction chemistry relies on an engineered DNA ligase and proprietary duplexed sequencing adapters that prevent chimeras, suppress dimer-formation (negating the need for size-selection), and enhance variant calling sensitivity. We adapted the workflow for both DNA and RNA applications and demonstrated efficacy using diverse sample types. To assess sensitivity, we created libraries with varied inputs using mixtures of Genome in a Bottle gDNA (NA12878 and NA24385) and performed hybrid capture using a 52 kb custom panel targeting: single nucleotide variants (SNVs), copy number variants (CNVs), and gene fusions. When compared to commercially available methods, our approach yielded a 1.5- to 4-fold increase in library complexity with improved sensitivity to 0.25% variants using 1-25 ng of cfDNA, and 0.5% using 25-250 ng FFPE DNA. We also obtained 100% specificity using duplexed UMI correction, which removed all false-positive calls. RNA libraries were constructed from FFPE NGS reference standards to evaluate the detection of ALK, RET, ROS, NTRK1, and NTRK3 fusions and sequenced to an average target depth of 10,000X. Our method provides superior sensitivity and specificity for detection of low-frequency variants, even with highly degraded DNA. Citation Format: Ariel Royall, Ushati Das Chakravarty, Katharine Dilger, Manqing Hong, Kevin Lai, Kristina Giorda, Keith Bryan, Yu Wang, Lynette Lewis, Scott Rose, Yu Zheng. Detection of low-frequency variants in highly degraded DNA and RNA samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3520.

For nonenveloped viruses such as Simian Virus 40, the mechanism used to translocate viral components across membranes is poorly understood. Previous results indicated that the minor structural proteins, VP2 and VP3, might act as membrane proteins during infection. Here, purified VP2 and VP3 were found to form pores in host cell membranes. To identify possible membrane domains, individual hydrophobic domains from VP2 and VP3 were expressed in a model protein and tested for their ability to integrate into membranes. Several domains from the late proteins supported endoplasmic reticulum membrane insertion as transmembrane domains. Mutations in VP2 and VP3 were engineered that inhibited membrane insertion and pore formation. When these mutations were introduced into the viral genome, viral propagation was inhibited. This comprehensive approach revealed that the viroporin activity of VP2 and VP3 was inhibited by targeted disruptions of individual hydrophobic domains and the loss of membrane disruption activity impaired viral infection.

Background The human genome contains variants ranging in size from small single nucleotide polymorphisms (SNPs) to large structural variants (SVs). High-quality benchmark small variant calls for the pilot National Institute of Standards and Technology (NIST) Reference Material (NA12878) have been developed by the Genome in a Bottle Consortium, but no similar high-quality benchmark SV calls exist for this genome. Since SV callers output highly discordant results, we developed methods to combine multiple forms of evidence from multiple sequencing technologies to classify candidate SVs into likely true or false positives. Our method (svclassify) calculates annotations from one or more aligned bam files from many high-throughput sequencing technologies, and then builds a one-class model using these annotations to classify candidate SVs as likely true or false positives. Results We first used pedigree analysis to develop a set of high-confidence breakpoint-resolved large deletions. We then used svclassify to cluster and classify these deletions as well as a set of high-confidence deletions from the 1000 Genomes Project and a set of breakpoint-resolved complex insertions from Spiral Genetics. We find that likely SVs cluster separately from likely non-SVs based on our annotations, and that the SVs cluster into different types of deletions. We then developed a supervised one-class classification method that uses a training set of random non-SV regions to determine whether candidate SVs have abnormal annotations different from most of the genome. To test this classification method, we use our pedigree-based breakpoint-resolved SVs, SVs validated by the 1000 Genomes Project, and assembly-based breakpoint-resolved insertions, along with semi-automated visualization using svviz. Conclusions We find that candidate SVs with high scores from multiple technologies have high concordance with PCR validation and an orthogonal consensus method MetaSV (99.7 % concordant), and candidate SVs with low scores are questionable. We distribute a set of 2676 high-confidence deletions and 68 high-confidence insertions with high svclassify scores from these call sets for benchmarking SV callers. We expect these methods to be particularly useful for establishing high-confidence SV calls for benchmark samples that have been characterized by multiple technologies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2366-2) contains supplementary material, which is available to authorized users.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

Simian virus 40 (SV40) appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection to aid in virus dissemination. To investigate the contribution of VP4 to cell lysis, VP4 was expressed in mammalian cells where it was predominantly observed along the nuclear periphery. The integrity of the nuclear envelope was compromised in these cells, resulting in the mislocalization of a soluble nuclear marker. Using assays that involved the cellular expression of VP4 or the treatment of cells with purified VP4, we found that the central hydrophobic domain and a proximal C-terminal nuclear localization signal of VP4 were required for (i) cytolysis associated with prolonged expression; (ii) nuclear envelope accumulation; and (iii) disruption of the nuclear, red blood cell, or host cell membranes. Furthermore, a conserved proline within the hydrophobic domain was required for membrane perforation, suggesting that this residue was crucial for VP4 cytolytic activity. These results indicate that VP4 forms pores in the nuclear membrane leading to lysis and virus release.

Introduction: Molecular diagnostics and precise personalized care continue to increase the sensitivity and specificity requirements for detecting low (~5%) to ultra-low (4,000 false positives, resulting in a positive predictive value (PPV) of 90% detection sensitivity was achieved for genomic and cell-free DNA samples with 0 false positives, resulting in 100% PPV, a >40-fold error suppression. For FFPE DNA samples, the duplex adapters provided >30-fold PPV improvement compared to standard adapters. Conclusions: The duplex UMI adapters eliminate background NGS errors by collapsing duplicated reads using their unique tags. This leads to unprecedented accuracy in detecting true low-frequency variants, regardless of the input DNA source. Such advances are key to refining diagnostics and improving precision cancer care. Citation Format: Jiashi Wang, Kevin Lai, Madelyn Light, Layla Katiraee, Kristina Giorda, Mirna Jarosz, Yun Bao, Criss Walworth, David Kupec, Caifu Chen. Highly efficient duplex DNA tagging strategy improves accuracy of detecting ultra-low-frequency mutations through consensus read reconstruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 418.

Complete gene knockouts are highly informative about gene function. We exome sequenced 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous likely loss of function (rhLOF) genotypes predicted to disrupt (knockout) 781 genes. Based on depletion of rhLOF genotypes, we estimate that 13.6% of knockouts are incompatible with adult life, finding on average 1.6 heterozygous recessive lethal LOF variants per adult. Linking to lifelong health records, we observed no association of rhLOF genotypes with prescription- or doctor-consultation rate, and no disease-related phenotypes in 33 of 42 individuals with rhLOF genotypes in recessive Mendelian disease genes. Phased genome sequencing of a healthy PRDM9 knockout mother, her child and controls, showed meiotic recombination sites localised away from PRDM9-dependent hotspots, demonstrating PRDM9 redundancy in humans.

Accurate genome-wide copy number variation (CNV) analysis is critical for disease and cancer research. Current approaches for CNV analysis include fluorescence in situ hybridization (FISH), array comparative genomic hybridization (array CGH), and SNP arrays. Unfortunately, these methods are not sensitive enough for real world cancer samples because of tumor ploidy, purity and heterogeneity. NGS-based targeted sequencing is increasingly being used for CNV analysis due to throughput, coverage, cost, and sample input requirements. For CNV analysis, detection power is improved by combining both read depth and SNP allele frequency analysis, particularly for copy-neutral events such as loss of heterozygosity. A custom xGen Lockdown CNV backbone panel was developed for broad, uniform genome coverage and to enrich for population-based SNPs. We demonstrate use of the panel as an addition to the xGen Exome Research Panel and a custom cancer focused panel. Downstream analysis incorporates both read depth and observed minor allele frequencies to determine CNVs with enhanced sensitivity. To increase the resolution for large-scale alterations of chromosome 7, a hot-spot for disease-associated CNVs, probe density was increased 6 fold. A known standard, NA12878, was used to validate the panel’s ability to detect heterozygous SNPs with high confidence. In addition, mixtures of cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were tested with varying levels of background copy-neutral genomic DNA. The sensitivity and specificity of the panel to detect CNV and LOH events with was assessed using deep exome and Affymetrix SNP array data. The ability to detect copy number alterations with high resolution and accuracy would be a valuable resource for disease and cancer research. Citation Format: Jiashi Wang, Kristina Giorda, Zhongwu Lai, Daniel Stetson, Mirna Jarosz. Whole genome copy number variation analysis using a SNP-focused targeted sequencing panel for tumor analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 397. doi:10.1158/1538-7445.AM2017-397

Neural tissue differentiated and cultured from patient-derived stem cells is expected to revolutionize the treatment of patients with brain and spinal injuries and diseases. Critical for these cellular therapies is accurate control and monitoring of differentiation but current methods for such cell typing are limited to qPCR and immunocytochemisty (ICC) which is not sufficient to discriminate between the numerous (likely >100,000) possible neural cell-types. RNA(transcriptome aka RNA-Seq) profiling permits the characterization and discovery of much-needed novel markers. To define the temporal transcriptional signature of neural stem cells, cultured human embryonic stem cells (H9) were compared to induced neural stem cells (NSCs) at d0, d7 and d14. ICC was performed on the putative NSC pools at d7 and d14 for markers of pluripotency (Oct4) and neural differentiation (nestin, Sox1, and Pax6) and H9 cells were stained on d14 for markers of pluripotency (Oct4 and SSEA4). Total RNA was isolated over the time c...

Dendritic cell (DC) lineages coordinate immune system activity through functional specialization. Irf4, a transcription factor, is required for CD11b+ DC lineage development from bone marrow stem cells and has been implicated in multiple inflammatory diseases such as asthma. The epigenetic consequences of immune specialization in CD11b+ DCs and relation to inflammatory diseases remain largely unexplored. One reason for this lack of genomic analysis stems from the difficulty of using highly purified, typically limited populations of cells in ChIP-seq assays. A robust ChIP protocol was developed using limited amounts of K562 cells as a benchmark study. ChIP-Seq analysis of the transcription factor CTCF or histone modifications strongly correlated with ENCODE data sets. This methodology was used to compare ChIP-seq analysis of Irf4 genomic binding sites using flow sorted populations of 1, 3, 5, or 25 million CD11b+ lineage DCs. Very similar quality scores and quantitative results comparing Irf4 ChIP-seq from...

Viruses are intracellular parasites that must access the host cell machinery to propagate. Viruses hijack the host cell machinery to help with entry, replication, packaging, and release of progeny to infect new cells. To carry out these diverse functions, viruses often transform the cellular environment using viroporins, a growing class of viral-encoded membrane proteins that promote viral proliferation. Viroporins modify the integrity of host membranes, thereby stimulating the maturation of viral infection, and are critical for virus production and dissemination. Significant advances in molecular and cell biological approaches have helped to uncover some of the roles that viroporins serve in the various stages of the viral life cycle. In this study, the ability of viroporins to modify the cellular environment will be discussed, with particular emphasis on their role in the stepwise progression of the viral life cycle.

Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results revealed that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of 1-5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.

Studies have shown that somatic structural variation (SV) plays a key role in the oncogenic process. Traditionally SVs in the cancer genome have been detected using low resolution cytogenetic approaches, such as FISH, or microarray-based techniques. More recently, next-generation sequencing (NGS)-based technologies have been employed to detect SVs, including indels and translocations. However, both short- and long-read NGS-based approaches are limited in their ability to accurately identify SV events and delineate their breakpoints due to the limitations inherent in assembly of billions of short-read sequences across a heterogeneous cancer sample, as well as the costly and burdensome laboratory infrastructure associated with long-read sequencers. We utilized a novel technology that combines microfluidics and molecular barcoding to generate libraries that are sequenced with an Illumina system. Open-source bioinformatics software produces linked-reads that maintain long-range information and single molecule sensitivity. Cell lines and cancer samples were obtained from commercial sources, and genomic DNA was extracted. DNA sample indexing and partitioning was performed using the 10X Genomicx GemCode instrument. One ng of sample DNA was used as input for each reaction, and DNA molecules were partitioned into droplets to fragment the DNA and introduce molecular barcodes. Following barcoding, droplets were fractured, and library DNA was purified and sequenced on Illumina sequencers. The GemCode Long Ranger software suite was used to map sequencing reads back to original long molecules of DNA, generating reads linked to partition barcodes. Thus we can generate phased sequences covering many 10's to 100's of kilobases. We first benchmarked the ability to call multiple SV types using a well-characterized germline HapMap sample (NA12878) as well as two recently characterized haploid hydatidiform moles (CHM1 and CHM13) that have been studied with multiple orthogonal technologies. Regions with evidence for structural variation were reassembled into distinct haplotypes. The barcode information allowed us to both phase the structural variants we detected and disambiguate calls within highly repetitive regions, such as segmental duplications. We demonstrated high concordance with alternative approaches across all major classes of SVs, including long insertions and deletions as well as copy-neutral events. In cancer cell lines, we detected well-annotated gene fusions, such as the EML4/ALK and ALK/PTPN3 fusions in the lung cancer cell line NCI-H2228, and the SLC26A/PRKAR2A fusion in the triple negative breast cancer cell line HCC38. Citation Format: Sofia Kyriazopoulou-Panagiotopoulou, Patrick Marks, Haynes Heaton, Heather Ordonez, Kristina Giorda, Cassandra Jabara, Billy Lau, John M. Bell, Michael Schnall-Levin, Hanlee P. Ji. Linked-Reads enable detailed, phased resolution of structural variation in the cancer genome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3602.

Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsulated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ∼3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release., Author Summary Viruses exploit host cells for their propagation. Once an adequate number of viral particles have been assembled within the cell through the aid of cellular machinery of the host cell, the virus must be released from the cell for the virus to spread. For nonenveloped viruses or viruses that are solely encapsulated by a protein shell, this step most commonly involves the perforation of cellular membranes resulting in the lysis or death of the host cell. The mechanism for how this key terminal step in the viral life cycle is performed is poorly understood. We demonstrated that for the model nonenveloped virus SV40, the newly discovered virally encoded protein, termed VP4, perforates membranes by forming pores with a diameter of ∼3 nm in host cell membranes. While these pores are not of a sufficient size to provide a conduit that permits the movement of the virus through the membrane, they support membrane destabilization that leads to the disintegration of the membrane of the host cell and viral release.

We have used a new platform from 10X Genomics to obtain ultra-deep and long-range exome sequencing data from 1ng of cancer cell line DNA. While traditional Whole Exome Sequencing (WES) methods have inputs of ∼50-500ng and lack long-range data, we demonstrate that the 10X platform can successfully obtain both gene fusion and haplotype phasing information along with standard SNP and indel variant-calling from WES on an Illumina HiSeq using just 1ng of DNA. The 10X platform massively partitions long DNA into >100,000 individual reactions. Each partition generates sequencing libraries with a unique barcode; the integrated downstream analysis platform uses the barcodes to map Illumina's short reads back to original long DNA fragments. Because the barcoding is performed prior to exome enrichment, the long-range information conveyed by the barcodes is retained even after standard hybrid capture enrichment. Powerful algorithms are implemented using a custom informatics toolset that is self-contained and easy to install, and the resulting structural variant calls can be easily visualized and explored using 10X's haplotype-aware genome browser. We used the 10X platform to analyze cancer cell lines with previously reported gene fusions. In all cases described, the fusion breakpoints were in introns of > 5.9kbp, and would therefore not be detectable with standard WES. In lung cancer cell line NCI-H2228, we called two annotated fusions, EML4/ALK, and ALK/PTPN3. Two fusion genes (MCHR2/ANO4, and CR627240/SACS) were identified in glioblastoma cell line U-87MG, and at least four (CD44/FGFR2, PVT1/PDHX, PVT1/ATE1, PVT1/PPAPDC1A) in gastric cancer cell line SNU-16. Long-range information provided by the technology also enables haplotype phasing of exome samples. Our algorithms phased the vast majority of SNPs within genes, with phase blocks up to 1.25Mbp. We used this haplotype information to phase somatic structural variants, including large-scale deletions and amplifications. To achieve the sensitivity required to call somatic mutations from low purity tumor specimens, it is necessary to be able to attain high coverage depth. This can be a significant challenge for low inputs; however we demonstrate duplicate-removed depth of >200x from inputs as low as 1ng, and sensitivity and precision for variant calling from 1ng libraries that is equivalent to that from ligation-based libraries prepared from 200ng of DNA. It is becoming increasingly apparent that a complete understanding of tumor genomic profiles requires the ability to detect structural variation and phasing information from low input and high coverage sequencing libraries. With this study we demonstrate the ability of the 10X platform to enable such applications from ultra-deep targeted sequencing data using only 1ng of DNA. Citation Format: Mirna Jarosz, Michael Schnall-Levin, Grace X. Y. Zheng, Patrick Marks, Sofia Kyriazopoulou-Panagiotopoulou, Patrice Mudivarti, Kristina Giorda. Using 1ng of DNA to detect haplotype phasing and gene fusions from whole exome sequencing of cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4742. doi:10.1158/1538-7445.AM2015-4742

Cancer genomes contain multiple types of genetic aberrations that include mutations, deletions, copy number variants and chromosomal rearrangements. Despite advances in next generation sequencing, it remains a major challenge to delineate many of these somatic genomic alterations because of intrinsic complexity of cancer genomes. Haplotyping involves the assignment of genetic variants such as mutations and structural variants to specific segment of homologous chromosomes. Experimentally determined phasing of cancer genomes offers an opportunity to resolve complex genomic structures such as somatic rearrangements, aneuploidy composition and ongoing evolutionary changes. However, contiguous phasing of cancer genomes on a megabase (Mb) scale remains difficult to achieve with sequencing-based approaches. In this proof-of-concept study, we experimentally determined Mb-scale haplotypes of primary tumor samples via whole genome sequencing. To generate haplotypes, we employed an automated instrument that partitions long DNA fragments into hundreds of thousands of reactions, each of which incorporates a unique, nonrandom barcode into indexed sequencing libraries. Given need to amplify from sparse numbers of molecules and the high efficiency of the automated sequencing library construction process, the DNA requirements for each sample are less than 5 ng. We sequenced the genomes of primary colorectal cancer samples and their matched normal diploid DNA with an Illumina sequencer. We used the single nucleotide variants to generate Mb-scale haplotype blocks (N50 of 1.2 Mb) with phased haplotype block size of up to 11.3 Mb. We were able to delineate cancer genome haplotypes that cover allelic imbalances, copy number variations such as deletions and other genomic instability events. Structural variants were identified in the context of their position in specific chromosome homologues. Thus, we improved the characterization of somatic genetic aberrations using contiguity mapping and cancer genome haplotypes in the context of whole cancer genome sequencing. Overall, we demonstrated the feasibility and potential utility of conducting contiguous phased haplotypes in whole cancer genome sequencing from primary tumor samples. Citation Format: Billy Lau, John M. Bell, Michael Schnall-Levin, Mirna Jarosz, Erik Hopmans, Christina M. Wood, Grace X. Zheng, Kristina Giorda, Hanlee P. Ji. Megabase-scale phased haplotypes of genetic aberrations from whole cancer genome sequencing of primary colorectal tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4882. doi:10.1158/1538-7445.AM2015-4882