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Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin lymphoma and remains incurable in ~40% of patients. Coding-genome sequencing efforts identified several genes/pathways altered in this disease, including new potential therapeutic targets(1–5). However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers (SEs) are highly and specifically hypermutated in 92% of DLBCL samples, display signatures of activation-induced cytidine deaminase (AID) activity, and are linked to genes encoding B-cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated SEs linked to the BCL6, BCL2, and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (BCL6) and the steroid-receptor NR3C1 (BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA-binding, downregulated target gene expression, and led to the counter-selection of cells harboring corrected alleles, indicating oncogenic dependency on the SE mutations. This pervasive SE mutational mechanism reveals a novel major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

Long-range oncogenic enhancers play an important role in cancer. Yet, whether similar regulation of tumor suppressor genes is relevant remains unclear. Loss of expression of PTEN is associated with the pathogenesis of various cancers, including T-cell acute lymphoblastic leukemia (T-ALL). Here, we identify a highly conserved distal enhancer (PE) that interacts with the PTEN promoter in multiple hematopoietic populations, including T cells, and acts as a hub of relevant transcription factors in T-ALL. Consistently, loss of PE leads to reduced PTEN levels in T-ALL cells. Moreover, PE-null mice show reduced Pten levels in thymocytes and accelerated development of NOTCH1-induced T-ALL. Furthermore, secondary loss of PE in established leukemias leads to accelerated progression and a gene expression signature driven by Pten loss. Finally, we uncovered recurrent deletions encompassing PE in T-ALL, which are associated with decreased PTEN levels. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in cancer.Significance:Here, we identify a PTEN enhancer that is recurrently deleted in patients with T-ALL. Loss of this enhancer leads to reduced PTEN levels in T cells together with accelerated generation and progression of NOTCH1-induced leukemia in vivo. These results uncover long-range regulation of tumor suppressor genes as a relevant mechanism in cancer.This article is highlighted in the In This Issue feature, p. 1

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Long-range oncogenic enhancers play an important role in cancer. Yet, whether similar regulation of tumor suppressor genes is relevant remains unclear. Loss of expression of PTEN is associated with the pathogenesis of various cancers, including T-cell acute lymphoblastic leukemia (T-ALL). Here, we identify a highly conserved distal enhancer (PE) that interacts with the PTEN promoter in multiple hematopoietic populations, including T cells, and acts as a hub of relevant transcription factors in T-ALL. Consistently, loss of PE leads to reduced PTEN levels in T-ALL cells. Moreover, PE-null mice show reduced Pten levels in thymocytes and accelerated development of NOTCH1-induced T-ALL. Furthermore, secondary loss of PE in established leukemias leads to accelerated progression and a gene expression signature driven by Pten loss. Finally, we uncovered recurrent deletions encompassing PE in T-ALL, which are associated with decreased PTEN levels. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in cancer.Significance:Here, we identify a PTEN enhancer that is recurrently deleted in patients with T-ALL. Loss of this enhancer leads to reduced PTEN levels in T cells together with accelerated generation and progression of NOTCH1-induced leukemia in vivo. These results uncover long-range regulation of tumor suppressor genes as a relevant mechanism in cancer.This article is highlighted in the In This Issue feature, p. 1

Activating mutations in cytosolic 5′-nucleotidase II (NT5C2) are considered to drive relapse formation in acute lymphoblastic leukemia (ALL) by conferring purine analog resistance. To examine the clinical effects of NT5C2 mutations in relapsed ALL, we analyzed NT5C2 in 455 relapsed B-cell precursor ALL patients treated within the ALL-REZ BFM 2002 relapse trial using sequencing and sensitive allele-specific real-time polymerase chain reaction. We detected 110 NT5C2 mutations in 75 (16.5%) of 455 B-cell precursor ALL relapses. Two-thirds of relapses harbored subclonal mutations and only one-third harbored clonal mutations. Event-free survival after relapse was inferior in patients with relapses with clonal and subclonal NT5C2 mutations compared with those without (19% and 25% vs 53%, P < .001). However, subclonal, but not clonal, NT5C2 mutations were associated with reduced event-free survival in multivariable analysis (hazard ratio, 1.89; 95% confidence interval, 1.28-2.69; P = .001) and with an increased rate of nonresponse to relapse treatment (subclonal 32%, clonal 12%, wild type 9%, P < .001). Nevertheless, 27 (82%) of 33 subclonal NT5C2 mutations became undetectable at the time of nonresponse or second relapse, and in 10 (71%) of 14 patients subclonal NT5C2 mutations were undetectable already after relapse induction treatment. These results show that subclonal NT5C2 mutations define relapses associated with high risk of treatment failure in patients and at the same time emphasize that their role in outcome is complex and goes beyond mutant NT5C2 acting as a targetable driver during relapse progression. Sensitive, prospective identification of NT5C2 mutations is warranted to improve the understanding and treatment of this aggressive ALL relapse subtype.

Coding-genome sequencing efforts have identified several genes/pathways altered in Diffuse Large B-cell Lymphoma (DLBCL), including new potential therapeutic targets. However, the mutational contribution of the non-coding genome of DLBCL remains largely unexplored. To identify functionally relevant non-coding mutations targeting regulatory domains, we integrated enhancer (E)/super-enhancer (SE) identification, by ChIP-seq analysis of the hallmark H3K27 histone mark, with whole genome sequencing (WGS) and RNA-seq analysis in 29 DLBCL cell lines representative of the major DLBCL subtypes, along with germinal center (GC) B cells representing the normal counterpart of DLBCL. Following validation in 93 normal/tumor DLBCL biopsies, we found that active SEs are specifically hypermutated (≥3 somatic mutations/Kb) in 99% of DLBCLs, as compared to the same loci when not active as SE (in total, 159 hypermutated SEs, with at least 2/case). As evidence of oncogenic relevance, we have shown that the hypermutated SEs linked to the BCL6 and BCL2 proto-oncogenes prevent the binding and transcriptional downregulation by transcriptional repressors. Genetic correction of selected mutations using the CRISPR/Cas9 technology restored repressor DNA-binding, downregulated target gene expression, and led to the counter-selection of cells harboring corrected alleles, indicating oncogenic dependency from the SE mutations. A third recurrently hypermutated SE was the CXCR4-SE, affected in 19% of cases. CXCR4 encodes for a G-protein coupled chemokine receptor involved in cell migration within the GC, and is mutationally activated in 40% of Waldenström macrobulinemia (WM), a post-GC lymphoproliferative disease. We identified a mutational hotspot preventing the binding of the NR3C1 glucocorticoid receptor in 7.5% of DLBCL cases. Correction of the mutations in cell lines led to counter selection and reduced expression of CXCR4. Conversely, the introduction of a mutation disrupting NR3C1 binding in WT DLBCL cells increased CXCR4 expression. Together, these results are consistent with dependency of the mutant cells on the CXCR4-SE mutation, and confirm a direct link between SE mutations in this region and deregulated gene expression through escape from NR3C1-mediated suppression. In primary cases, mutations in the NR3C1 binding site of the CXCR4-SE were observed in the absence of NR3C1 coding mutations and correlated with increased CXCR4 transcript levels, as documented by RNA-seq. Together with the oncogenic role of CXCR4 in WM, these findings reveal this chemokine receptor as a potential oncogenic target in DLBCL. Collectively, these findings reveal a new layer of genetic alterations, which identifies novel mechanisms of dysregulation for known oncogenes, as well as new dysregulated genes and pathways, with implications for precision classification and therapeutic targeting of DLBCL. * Co-senior authors. Citation Format: Elodie Bal, Rahul Kumar, Mohammad Hadigol, Antony B. Holmes, Laura K. Hilton, Jui Wan Loh, Kostiantyn Dreval, Jasper Wong, Sofija Vlasevska, Clarissa Corinaldesi, Rajesh Kumar Soni Soni, Katia Basso, Ryan D. Morin, Hossein Khiabanian, Laura Pasqualucci, Riccardo Dalla-Favera. Pervasive hypermutation of super-enhancer regions dysregulates oncogene expression in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A12.

Advances in high-throughput methods have enabled the molecular characterization of leukemias and have improved our understanding of their clonal evolution from leukemogenesis in hematopoietic stem/progenitor cells to overt diagnosable disease. It has now been revealed that during leukemia’s development and progression, genetic alterations accumulate according to the principles of Darwinian evolution. Drug resistance often emerges from changes in evolutionary trajectories of disease through selection of subpopulations that have greater fitness under therapy. In this manuscript, we will review recent data on prevalence of highly branched evolutionary patterns in myeloid and lymphoid leukemias and discuss how different treatment strategies differentially shape leukemia’s clonal architecture. Increasing evidence on clinical impact of small pre-malignant clones prior to diagnosis and small resistant clones during treatment strongly suggests that highly sensitive experimental and mathematical models are necessary for accurate dissection of hematopoietic populations and robust identification of predictive markers for disease transformation and relapse.

Context: Ibrutinib inhibits BTK with the most typical response in CLL being partial remission (PR) with measurable minimal residual disease (MRD) in blood. Remission is usually maintained until development of genetically-driven resistance. Objective: Mechanism of adaptation and survival in MRD. Patients and Methods: Pre-treatment CLL cells and under-treatment MRD were systematically collected from 33 high-risk CLL patients. Samples were characterized by high-dimensional, single-cell flow cytometry; bulk genomic, transcriptomic, and chromatin accessibility profiling; and single-cell RNA-seq profiling. Results: The genetic composition of MRD remained constant across timepoints, indicating that ibrutinib did not have any impact in shaping its genomic diversity. Ibrutinib induced chromatin dynamics in MRD resulting in a predominantly closed state. Chromatin regions that became more accessible under ibrutinib were enriched of binding sites for transcription factor that lay downstream ERK signaling. Regions that turned into a less accessible state were enriched for the binding sites of TF emanating from the BCR. At the transcriptomic level, most of the differentially expressed genes between pre- and post-treatment samples were downregulated in MRD, while only a fraction was upregulated. IL4, NF-kB, and metabolism and proliferation signatures were downregulated in MRD, while MAPK and RAS signatures were upregulated. CLL spleen samples from TCL1 mice under ibrutinib treatment showed an upregulation of MAPK pathway genes compared to untreated mice. Single-cell transcriptomes revealed a distinct post-treatment cell population driven by MAPK activity that functionally pre-existed in a fraction of CLL cells of baseline samples before ibrutinib start. At the signaling level, ibrutinib had no effect on the integrity of RAS-BRAF-MAPK-ERK pathway protein expression upon crosslinking of the BCR with anti-IgM but not after stimulation of TLR9 or CD40. Functional validation on primary samples by western blotting shows that RAS/ERK can be activated by BCR stimulation, irrespective of ibrutinib treatment in ex vivo experiments. Pharmacological inhibition of MEK (trametinib) and ERK (ulixertinib) synergized with ibrutinib, leading to decreased cell viability. Conclusions: MRD under ibrutinib adapts its phenotype in an epigenetic way to maintain functional competence of BCR signaling via the MAPK pathway, which turned to be a vulnerability of MRD persisting under ibrutinib.

Additional file 5: Figure S3. Potential functional impact of pMEIs. A) functional annotation; B) abundance of pMEIs in different chromatin states. Chromatin state profiles (Y-axis) from nine cell lines (X-axis) were obtained from ChromHMM [42]. For each chromatin state, the normalized number of pMEIs is shown. Chromatin States: 1 - Active Promoter, 2 - Weak Promoter, 3 - Inactive/poised Promoter, 4 - Strong enhancer, 5 - Strong enhancer, 6 - Weak/poised enhancer, 7 - Weak/poised enhancer, 8 – Insulator, 9 - Transcriptional transition, 10 - Transcriptional elongation, 11 - Weak transcribed, 12 - Polycomb-repressed, 13 - Heterochromatin; low signal, 14 - Repetitive/CNV, 15 - Repetitive/CNV.

Additional file 3: Figure S1. Computational pipeline for ME-Scan analysis.

Additional file 6: Figure S4. Locus-specific PCR validation. A) Alu_CDS1; B) L1_CDS2, L1_CDS4, L1_CDS5, L1_CDS7; C) Sequence of the Alu_CDS1 locus. Each individual ID is labelled on the top of the lane. Ladder, 100 bp ladder. The main ladder bands (500 bps, 1000 bps) are labelled. The expected empty allele (i.e. no insertion) size is labelled on the top of the lane. For L1 loci, three PCR reactions were performed. Left panel: E: outside forward + reverse primer, I: internal L1_1 + outside forward primer; right panel: internal L1_2 + outside forward primer. The expected internal + external size is around 500 bps although it varies because of the variable poly(A) length of the L1 insertion. The expected internal + outside primer amplification product is indicated by black arrow. Because we were unable to amplify the full insertion allele by the external primers, we did not validate both ends of the L1 insertion by Sanger sequencing. For the Sanger sequencing result Alu_CDS1, the AluY insertion is highlighted in green, the target site duplications are in red, and the potential endonuclease cutting site is underlined.

Peritoneal carcinomatosis (PC) present a ubiquitous clinical conundrum in all intra-abdominal malignancies. Via functional and transcriptomic experiments of ascites-treated PC cells, we identify STAT3 as a key signaling pathway. Integrative analysis of publicly available databases and correlation with clinical cohorts (n = 7,359) reveal putative clinically significant activating ligands of STAT3 signaling. We further validate a 3-biomarker prognostic panel in ascites independent of clinical covariates in a prospective study (n = 149). Via single-cell sequencing experiments, we uncover that PAI-1, a key component of the prognostic biomarker panel, is largely secreted by fibroblasts and mesothelial cells. Molecular stratification of ascites using PAI-1 levels and STAT3 activation in ascites-treated cells highlight a therapeutic opportunity based on a phenomenon of paracrine addiction. These results are recapitulated in patient-derived ascites-dependent xenografts. Here, we demonstrate therapeutic proof of concept of direct ligand inhibition of a prognostic target within an enclosed biological space.

Background Mobile elements are ubiquitous components of mammalian genomes and constitute more than half of the human genome. Polymorphic mobile element insertions (pMEIs) are a major source of human genomic variation and are gaining research interest because of their involvement in gene expression regulation, genome integrity, and disease. Results Building on our previous Mobile Element Scanning (ME-Scan) protocols, we developed an integrated ME-Scan protocol to identify three major active families of human mobile elements, AluYb, L1HS, and SVA. This approach selectively amplifies insertion sites of currently active retrotransposons for Illumina sequencing. By pooling the libraries together, we can identify pMEIs from all three mobile element families in one sequencing run. To demonstrate the utility of the new ME-Scan protocol, we sequenced 12 human parent-offspring trios. Our results showed high sensitivity (> 90%) and accuracy (> 95%) of the protocol for identifying pMEIs in the human genome. In addition, we also tested the feasibility of identifying somatic insertions using the protocol. Conclusions The integrated ME-Scan protocol is a cost-effective way to identify novel pMEIs in the human genome. In addition, by developing the protocol to detect three mobile element families, we demonstrate the flexibility of the ME-Scan protocol. We present instructions for the library design, a sequencing protocol, and a computational pipeline for downstream analyses as a complete framework that will allow researchers to easily adapt the ME-Scan protocol to their own projects in other genomes.

Summary Clinical sequencing aims to identify somatic mutations in cancer cells for accurate diagnosis and treatment. However, most widely used clinical assays lack patient-matched control DNA and additional analysis is needed to distinguish somatic and unfiltered germline variants. Such computational analyses require accurate assessment of tumor cell content in individual specimens. Histological estimates often do not corroborate with results from computational methods that are primarily designed for normal–tumor matched data and can be confounded by genomic heterogeneity and presence of sub-clonal mutations. Allele-frequency-based imputation of tumor (All-FIT) is an iterative weighted least square method to estimate specimen tumor purity based on the allele frequencies of variants detected in high-depth, targeted, clinical sequencing data. Using simulated and clinical data, we demonstrate All-FIT’s accuracy and improved performance against leading computational approaches, highlighting the importance of interpreting purity estimates based on expected biology of tumors. Availability and implementation Freely available at http://software.khiabanian-lab.org. Supplementary information Supplementary data are available at Bioinformatics online.

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect.

Despite intensive chemotherapy, 20% of pediatric acute lymphoblastic leukemia (ALL) patients fail to achieve a complete remission or relapse after intensified chemotherapy, making relapse and resistance to therapy the most significant challenge in the treatment of this disease. We have shown that NT5C2 mutations resulting in deregulated enzymatic activity are present in 10% of relapsed B-precursor ALL and 20% of relapsed T-ALL cases. These genetic alterations abrogate the activity of an intramolecular switch-off mechanism resulting in increased nucleotidase activity. NT5C2 metabolizes and clears the activated forms of 6-mercaptopurine (6-MP), a critical drug in the treatment of ALL. As a result, activating mutations in the NT5C2 cytosolic nucleotidase gene are characteristically associated with early relapsed leukemia and progression under therapy and confer resistance to 6-mercaptopurine chemotherapy. A saturated mutagenesis positive selection screen for all possible NT5C2 variants (10640 alleles) conferring resistance to 6-MP recovers mutational hotspots involving recurrent relapse-associated mutations and identifies new residues and protein domains involved in NT5C2 regulation whose inactivation drives resistance to 6-MP. Moreover, a genome wide CRISPR screen analysis of 6-MP-gene interactions in NT5C2 wild type REH ALL cells identifies NT5C2 as the most prominent hit whose inactivation increases sensitivity to 6-MP. Taken together, these results point to heretofore uncharacterized mechanisms of NT5C2 regulation and support a role for wild type NT5C2 in 6-MP metabolism and inactivation. Thus post-translational modifications enhancing NT5C2 activity could attenuate the therapeutic activity of 6-MP in NT5C2 wild type cells as non-mutational mechanism of 6-MP resistance. Mass spectrometry analysis of NT5C2 protein revealed the presence of two NT5C2 acetylations and two NT5C2 phosphorylation sites. Functional analyses of these modifications revealed increased nucleotidase activity for the NT5C2 S502D phospho-mimic mutation. Consistently, expression of NT5C2 S502D in leukemia lymphoblasts induced resistance to 6-MP. Importantly, western blot analysis revealed increased NT5C2 S502 phosphorylation in relapsed leukemia xenograft samples compared with matched diagnosis derived ALL xenografts. Crystal structure analysis of NT5C2 recombinant protein supports that S502 engages D229 in the partner protomer of the NT5C2 dimer. Structure-function analysis of S502D, S502A and D229A mutants targeting this interaction supports the disruption of S502-D229 interaction via S502 phosphorylation as mechanism of NT5C2 activation. In all, these results highlight a prominent role for wild type NT5C2 activity in the detoxification of 6-MP in ALL lymphoblasts, identifies NT5C2 S502 phosphorylation as a prevalent non-genetic mechanism of NT5C2 activation at relapse and provides novel insight on the mechanisms of NT5C2 regulation of relevance for the therapeutic targeting of NT5C2 in relapsed ALL. Disclosures No relevant conflicts of interest to declare.

Background: Patients with unfavorable AML have an estimated 3-year overall survival (OS) of < 15% with standard chemotherapy treatment. The recommendation for consolidative allogeneic transplant can be challenging and is usually an individualized assessment. Accurate and sensitive genomic data may improve the decision making process. Detailed mutational analyses using myeloid-specific gene assays have shown to refine disease prognostication from standard six-gene mutational analysis (Gerstung et al. Nature Genetics2017, Dohner et al. Blood2017). Several studies using mutation analysis for assessing minimal residual disease (MRD) have been performed. Specifically, in two large studies, isolated presence of DNMT3A, TET2, or ASXL1mutations (associated with clonal hematopoiesis) was not prognostic but detection of other mutations, independent of pathogenicity, at >2.5% predicted relapse, relapse-free survival, and overall survival (Jongen-Lavrencic et al. JEJM2018, Morita et al. JCO2018). Of note, flow cytometry was also used to detect minimal residual disease (MRD), and at a sensitivity of 0.1% provided independent prognostic value; however even when used in combination, nearly 25% of relapses were not predicted. At Rutgers, we utilize deep DNA sequencing and real-time multiplex PCR assays to detect mutant alleles in the presence of 10,000 wild-type fragments. Here, we hypothesize that detailed analysis of available mutational data and detection of mutations at allele frequencies Methods: We recruited 99 patients with newly diagnosed high-risk MDS and/or AML.Each patient had standard myeloid mutation profiles obtained at diagnosis (pre-treatment) and on the 'day 30' recovery bone marrow. Patient characteristics and bone marrow biopsy results were obtained including standard cytogenetics and conventional molecular studies. Additional and contemporaneous analyses were performed for 18 patients, diagnosed and treated from February 2017 through July 2019,utilizinga 49-gene panel (RainDance Technologies) on Illumina MiSeq at high depth (2,000x-20,000x). We identified all sites different from the reference using an inclusive variant caller and used a Bayesian approach to detect true mutations against mutation-specific background error at >0.1% allele frequencies. We tested the variants against previously sequenced non-malignant samples and guided the analysis by detected mutations in longitudinal data (Hadigol et al. BMC Bioinformatics2018, Rabadan et al. J Stat Phys2018). Results:The median age of patients with deep-sequencing data was 55 years (range, 26-73 [n=4 male; n=14 female]). There were 14 patients with AML and 4 patients with high-risk MDS. Sixty-one percent of patients received standard 7+3 induction therapy and 14 patients received consolidative allogeneic stem cell transplant (SCT), 93% of whom received it at first complete remission (CR1). With the median follow-up of 19 months (range, 12-27), the median time to relapse in 5 patients (28%) was 13 months (range, 8-15). In the relapsed patients, there was a median of 1 mutation (range, 0-2) reported by conventional variant calling methods at CR1. However, Bayesian deep-sequencing analysis identified additional non-synonymous coding mutations (range, 1-4) at allele frequencies Conclusion: Our approach identified new and relevant mutations that were not reported with conventional genomic analysis at a crucial time point in the measurement of treatment response in AML and high-risk MDS. Data such as these may refine prognostication and ultimately be used as to help guide further treatment decision making for patients. Continued and larger prospective analyses with novel deep-sequencing technology are needed to better inform outcomes for AML and high-risk MDS patients. Disclosures No relevant conflicts of interest to declare.

Background Mobile element insertions are a major source of human genomic variation. SVA (SINE-R/VNTR/Alu) is the youngest retrotransposon family in the human genome and a number of diseases are known to be caused by SVA insertions. However, inter-individual genomic variations generated by SVA insertions and their impacts have not been studied extensively due to the difficulty in identifying polymorphic SVA insertions. Results To systematically identify SVA insertions at the population level and assess their genomic impact, we developed a mobile element scanning (ME-Scan) protocol we called ME-Scan-SVA. Using a nested SVA-specific PCR enrichment method, ME-Scan-SVA selectively amplify the 5′ end of SVA elements and their flanking genomic regions. To demonstrate the utility of the protocol, we constructed and sequenced a ME-Scan-SVA library of 21 individuals and analyzed the data using a new analysis pipeline designed for the protocol. Overall, the method achieved high SVA-specificity and over >90 % of the sequenced reads are from SVA insertions. The method also had high sensitivity (>90 %) for fixed SVA insertions that contain the SVA-specific primer-binding sites in the reference genome. Using candidate locus selection criteria that are expected to have a 90 % sensitivity, we identified 151 and 29 novel polymorphic SVA candidates under relaxed and stringent cutoffs, respectively (average 12 and 2 per individual). For six polymorphic SVAs that we were able to validate by PCR, the average individual genotype accuracy is 92 %, demonstrating a high accuracy of the computational genotype calling pipeline. Conclusions The new approach allows identifying novel SVA insertions using high-throughput sequencing. It is cost-effective and can be applied in large-scale population study. It also can be applied for detecting potential active SVA elements, and somatic SVA retrotransposition events in different tissues or developmental stages. Electronic supplementary material The online version of this article (doi:10.1186/s13100-016-0072-x) contains supplementary material, which is available to authorized users.