Formalin-Fixed, Paraffin-Embedded-Targeted Locus Capture: A Next-Generation Sequencing Technology for Accurate DNA-Based Gene Fusion Detection in Bone and Soft Tissue Tumors.

Chromosomal rearrangements are important drivers in cancer, and their robust detection is essential for diagnosis, prognosis, and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations, including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based next-generation sequencing method, formalin-fixed, paraffin-embedded-targeted locus capture (FFPE-TLC), has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. We evaluated the utility of FFPE-TLC in bone and soft tissue tumor diagnostics. FFPE-TLC sequencing was successfully applied on noncalcified and decalcified FFPE samples (n = 44) and control samples (n = 19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples, and none was identified in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed due to either detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors.

Background: Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors. Materials and methods: Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed. Results: TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse. Conclusion: We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.

Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia.

Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.

Targeted Locus Amplification and Haplotyping.

Targeted locus amplification (TLA) allows for the detection of all genetic variation (including structural variation) in a genomic region of interest. As TLA is based on proximity ligation, variants can be linked to each other, thereby enabling allelic phasing and the generation of haplotypes. This allows for the study of genetic variants in an allele-specific manner. Here, we provide a step-by-step protocol for TLA sample preparation and a complete bioinformatics pipeline for the allelic phasing of TLA data. Additionally, to illustrate the protocol, we show the ability of TLA to re-sequence and haplotype the complete cystic fibrosis transmembrane (CFTR) gene (> 200 kb in size) from patient-derived intestinal organoids.

Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing.

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Targeted locus amplification to detect molecular markers in mantle cell and follicular lymphoma.

Minimal residual disease (MRD) monitoring by PCR methods is a strong and standardized predictor of clinical outcome in mantle cell lymphoma (MCL) and follicular lymphoma (FL). However, about 20% of MCL and 40% of FL patients lack a reliable molecular marker, being thus not eligible for MRD studies. Recently, targeted locus amplification (TLA), a next-generation sequencing (NGS) method based on the physical proximity of DNA sequences for target selection, identified novel gene rearrangements in leukemia. The aim of this study was to test TLA in MCL and FL diagnostic samples lacking a classical, PCR-detectable, t(11; 14) MTC (BCL1/IGH), or t(14; 18) major breakpoint region and minor cluster region (BCL2/IGH) rearrangements. Overall, TLA was performed on 20 MCL bone marrow (BM) or peripheral blood (PB) primary samples and on 20 FL BM, identifying a novel BCL1 or BCL2/IGH breakpoint in 16 MCL and 8 FL patients (80% and 40%, respectively). These new breakpoints (named BCL1-TLA and BCL2-TLA) were validated by ASO primers design and compared as MRD markers to classical IGH rearrangements in eight MCL: overall, MRD results by BCL1-TLA were superimposable (R Pearson = 0.76) to the standardized IGH-based approach. Moreover, MRD by BCL2-TLA reached good sensitivity levels also in FL and was predictive of a primary refractory case. In conclusion, this study offers the proof of principle that TLA is a promising and reliable NGS-based technology for the identification of novel molecular markers, suitable for further MRD analysis in previously not traceable MCL and FL patients.

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next-Generation Sequencing Technologies for Cell Line Development.

Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next-generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single-nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high-level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.

Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis.

Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

Genetic Screening Test to Detect Translocations in Acute Leukemias by Use of Targeted Locus Amplification.

BACKGROUND: Over 500 translocations have been identified in acute leukemia. To detect them, most diagnostic laboratories use karyotyping, fluorescent in situ hybridization, and reverse transcription PCR. Targeted locus amplification (TLA), a technique using next-generation sequencing, now allows detection of the translocation partner of a specific gene, regardless of its chromosomal origin. We present a TLA multiplex assay as a potential first-tier screening test for detecting translocations in leukemia diagnostics. METHODS: The panel includes 17 genes involved in many translocations present in acute leukemias. Procedures were optimized by using a training set of cell line dilutions and 17 leukemia patient bone marrow samples and validated by using a test set of cell line dilutions and a further 19 patient bone marrow samples. Per gene, we determined if its region was involved in a translocation and, if so, the translocation partner. To balance sensitivity and specificity, we introduced a gray zone showing indeterminate translocation calls needing confirmation. We benchmarked our method against results from the 3 standard diagnostic tests. RESULTS: In patient samples passing QC, we achieved a concordance with benchmarking tests of 81% in the training set and 100% in the test set, after confirmation of 4 and nullification of 3 gray zone calls (in total). In cell line dilutions, we detected translocations in 10% aberrant cells at several genetic loci. CONCLUSIONS: Multiplex TLA shows promising results as an acute leukemia screening test. It can detect cryptic and other translocations in selected genes. Further optimization may make this assay suitable for diagnostic use.

X chromosome inactivation in a female carrier of a 1.28 Mb deletion encompassing the human X inactivation centre.

X chromosome inactivation (XCI) is a mechanism specifically initiated in female cells to silence one X chromosome, thereby equalizing the dose of X-linked gene products between male and female cells. XCI is regulated by a locus on the X chromosome termed the X-inactivation centre (XIC). Located within the XIC is XIST, which acts as a master regulator of XCI. During XCI, XIST is upregulated on the inactive X chromosome and chromosome-wide cis spreading of XIST leads to inactivation. In mouse, the Xic comprises Xist and all cis-regulatory elements and genes involved in Xist regulation. The activity of the XIC is regulated by trans-acting factors located elsewhere in the genome: X-encoded XCI activators positively regulating XCI, and autosomally encoded XCI inhibitors providing the threshold for XCI initiation. Whether human XCI is regulated through a similar mechanism, involving trans-regulatory factors acting on the XIC has remained elusive so far. Here, we describe a female individual with ovarian dysgenesis and a small X chromosomal deletion of the XIC. SNP-array and targeted locus amplification (TLA) analysis defined the deletion to a 1.28 megabase region, including XIST and all elements and genes that perform cis-regulatory functions in mouse XCI. Cells carrying this deletion still initiate XCI on the unaffected X chromosome, indicating that XCI can be initiated in the presence of only one XIC. Our results indicate that the trans-acting factors required for XCI initiation are located outside the deletion, providing evidence that the regulatory mechanisms of XCI are conserved between mouse and human.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.

Genetic and epigenetic features direct differential efficiency of Xist-mediated silencing at X-chromosomal and autosomal locations.

Xist is indispensable for X chromosome inactivation. However, how Xist RNA directs chromosome-wide silencing and why some regions are more efficiently silenced than others remains unknown. Here, we explore the function of Xist by inducing ectopic Xist expression from multiple different X-linked and autosomal loci in mouse aneuploid and female diploid embryonic stem cells in which Xist-mediated silencing does not lead to lethal functional monosomy. We show that ectopic Xist expression faithfully recapitulates endogenous X chromosome inactivation from any location on the X chromosome, whereas long-range silencing of autosomal genes is less efficient. Long interspersed elements facilitate inactivation of genes located far away from the Xist transcription locus, and genes escaping X chromosome inactivation show enrichment of CTCF on X chromosomal but not autosomal loci. Our findings highlight important genomic and epigenetic features acquired during sex chromosome evolution to facilitate an efficient X chromosome inactivation process.Xist RNA is required for X chromosome inactivation but it is not well understood how Xist silences some regions more efficiently than others. Here, the authors induce ectopic Xist expression from multiple different X-linked and autosomal loci in cells to explore Xist function.

Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping.

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification.

Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events.

Quantitative analysis of chromatin interaction changes upon a 4.3 Mb deletion at mouse 4E2.

BACKGROUND: Circular chromosome conformation capture (4C) has provided important insights into three dimensional (3D) genome organization and its critical impact on the regulation of gene expression. We developed a new quantitative framework based on polymer physics for the analysis of paired-end sequencing 4C (PE-4Cseq) data. We applied this strategy to the study of chromatin interaction changes upon a 4.3 Mb DNA deletion in mouse region 4E2. RESULTS: A significant number of differentially interacting regions (DIRs) and chromatin compaction changes were detected in the deletion chromosome compared to a wild-type (WT) control. Selected DIRs were validated by 3D DNA FISH experiments, demonstrating the robustness of our pipeline. Interestingly, significant overlaps of DIRs with CTCF/Smc1 binding sites and differentially expressed genes were observed. CONCLUSIONS: Altogether, our PE-4Cseq analysis pipeline provides a comprehensive characterization of DNA deletion effects on chromatin structure and function.

CTCF Binding Polarity Determines Chromatin Looping.

CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure.

Dynamics of gene silencing during X inactivation using allele-specific RNA-seq.

BACKGROUND: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. RESULTS: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. CONCLUSIONS: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome.

Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping.

Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.

Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization.

The relationship between 3D organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in mouse embryonic stem cells (ESCs), iPSCs, and fibroblasts and uncovered a pluripotency-specific genome organization that is gradually reestablished during reprogramming. Our data confirm that long-range chromatin interactions are primarily associated with the spatial segregation of open and closed chromatin, defining overall chromosome conformation. Additionally, we identified two further levels of genome organization in ESCs characterized by colocalization of regions with high pluripotency factor occupancy and strong enrichment for Polycomb proteins/H3K27me3, respectively. Underlining the independence of these networks and their functional relevance for genome organization, loss of the Polycomb protein Eed diminishes interactions between Polycomb-regulated regions without altering overarching chromosome conformation. Together, our data highlight a pluripotency-specific genome organization in which pluripotency factors such as Nanog and H3K27me3 occupy distinct nuclear spaces and reveal a role for cell-type-specific gene-regulatory networks in genome organization.

The pluripotent genome in three dimensions is shaped around pluripotency factors.

It is becoming increasingly clear that the shape of the genome importantly influences transcription regulation. Pluripotent stem cells such as embryonic stem cells were recently shown to organize their chromosomes into topological domains that are largely invariant between cell types. Here we combine chromatin conformation capture technologies with chromatin factor binding data to demonstrate that inactive chromatin is unusually disorganized in pluripotent stem-cell nuclei. We show that gene promoters engage in contacts between topological domains in a largely tissue-independent manner, whereas enhancers have a more tissue-restricted interaction profile. Notably, genomic clusters of pluripotency factor binding sites find each other very efficiently, in a manner that is strictly pluripotent-stem-cell-specific, dependent on the presence of Oct4 and Nanog protein and inducible after artificial recruitment of Nanog to a selected chromosomal site. We conclude that pluripotent stem cells have a unique higher-order genome structure shaped by pluripotency factors. We speculate that this interactome enhances the robustness of the pluripotent state.