BackgroundSequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information. To combine next generation sequencing technology with SRAP, Illumina's Solexa sequencing was used to sequence tagged SRAP PCR products.ResultsThree sets of SRAP primers and three sets of tagging primers were used in 77,568 SRAP PCR reactions and the same number of tagging PCR reactions respectively to produce a pooled sample for Illumina's Solexa sequencing. After sequencing, 1.28 GB of sequence with over 13 million paired-end sequences was obtained and used to match Solexa sequences with their corresponding SRAP markers and to integrate Solexa sequences on an ultradense genetic map. The ultradense genetic bin map with 465 bins was constructed using a recombinant inbred (RI) line mapping population in B. rapa. For this ultradense genetic bin map, 9,177 SRAP markers, 1,737 integrated unique Solexa paired-end sequences and 46 SSR markers representing 10,960 independent genetic loci were assembled and 141 unique Solexa paired-end sequences were matched with their corresponding SRAP markers. The genetic map in B. rapa was aligned with the previous ultradense genetic map in B. napus through common SRAP markers in these two species. Additionally, SSR markers were used to perform alignment of the current genetic map with other five genetic maps in B. rapa and B. napus.ConclusionWe used SRAP to construct an ultradense genetic map with 10,960 independent genetic loci in B. rapa that is the most saturated genetic map ever constructed in this species. Using next generation sequencing, we integrated 1,878 Solexa sequences on the genetic map. These integrated sequences will be used to assemble the scaffolds in the B. rapa genome. Additionally, this genetic map may be used for gene cloning and marker development in B. rapa and B. napus.

Background: Gene Fusion events are common occurrences in malignancies, and are frequently drivers of malignancy. FISH and qPCR are two methods often used for identifying highly prevalent gene fusions/translocations. However, these are single target assays, requiring a lot of effort and sample if multiple assays are needed for multiple targets like sarcoma. High-throughput parallel (NextGen) DNA and RNA sequencing are also in current use to detect and characterize gene fusions. RNA sequencing (RNAseq) has the advantage that multiple markers can be targeted at one time and RNA fusions are readily identified from their product transcripts. While many fusion calling algorithms exist for use on RNAseq data, sensitive fusion callers, needed for samples of low tumor content, often present high false positive rates. Further, there currently is no single variable or element in NGS data that can be used to filter out false positive calls by extant callers. Individual sensitive fusion callers may be considered weak predictors of gene fusions. Combining their results into a single fusion call involves evaluating many elements, which can be a time consuming and difficult manual task. In order to achieve higher accuracy in fusion calls than can be achieved using individual fusion callers, we have combined the results of multiple fusion callers by use of an ensemble learning approach based on random forest models. Our method selects the best group of callers from among several callers, and provides an algorithmic means of combining their results, presenting a metric that can be immediately interpreted as the probability that a called fusion is a true fusion call. Methods: Random forest models were generated with the randomForest package in R, and then tuned using the R caret package. Training data sets consisted of fusion calls deemed true by review and by orthogonal methods including PCR/Sanger sequencing and the commercial Archer™ fusion calling system. We present the results of training on calls made by five fusion callers Arriba, STAR-Fusion, FusionCatcher, deFuse, and Kallisto/pizzly. Logistic training variables (seen vs not seen by the fusion caller) were used for the five callers. Variables also included metrics for the magnitude and balance of coverage on either side of candidate fusion breakpoints reported by Arriba and STAR Fusion ("coverage balance") and a single metric consisting of the number of sequencing reads that cross the candidate breakpoint. The model was validated by 10-fold cross-validation on 598 fusion calls by the five callers. Results: The resulting model is superior to the simple strategy of requiring agreement by n of five callers, particularly with regard to specificity (Table 1). Also, "importance of variables," reported by randomForest, gauges the relative contribution of variables in the model. Here it shows that one caller, Kallisto\pizzly, does not contribute to the model (Table 2). Conclusion: Random Forest modeling provides a viable means of combining gene fusion call data from multiple callers into a single fusion calling tool with improved performance over simple combinations of fusion calls. An additional benefit is seen in that building and evaluating such models can guide the selection of fusion callers, thereby eliminating non-contributory calling methods and ensuring optimal utilization of computational resources. Disclosures Thomas: NeoGenomics,Inc.: Current Employment. Mou:NeoGenomics: Current Employment. Keeler:NeoGenomics: Current Employment. Magnan:NeoGenomics: Current Employment. Funari:NeoGenomics: Current Employment. Weiss:Merck: Other: Speaker; Bayer: Other: speaker; Genentech: Other: Speaker; NeoGenomics: Current Employment. Brown:NeoGenomics,Inc.: Current Employment. Agersborg:NeoGenomics: Current Employment.

Integrins play an important role in lymphocyte adhesion to cellular and extracellular components of their microenvironment. The regulation of such adhesion often involves changes in the functional state of the integrins rather than alterations in their expression levels. Although the functional basis for such transitions is unknown, a possible role for disulfide exchange might be postulated based on the observations that integrin function can be activated by bifunctional reducing agents or by Abs that react with areas adjacent to predicted long-range disulfide bonds in integrins. Recently, it has been reported that enzymes that catalyze disulfide exchanges such as protein disulfide isomerase (PDI) are present on the surface of lymphoid cells, raising the possibility that such enzymes might be involved in the control of lymphocyte adhesion. A number of inhibitors of PDI function were examined for their effects on integrin-mediated adherence of T cells. The results did not support role for PDI in the regulation of integrin function, as the inhibitors somatostatin A, tocinoic acid, dithiobisnitrobenzoic acid, and anti-PDI mAb did not interfere with adherence. However, one of the PDI inhibitors, bacitracin, selectively interfered with the β1 integrin-mediated adherence of lymphoid cells to collagen, fibronectin, laminin, and VCAM-1, and with α4β7-dependent adherence to fibronectin and to VCAM-1. In contrast, αvβ3- and αLβ2-mediated adherence were not inhibited. Thus, it appears that bacitracin may be a selective inhibitor of β1 and β7 integrin functions by an as yet unknown mechanism.

Background: To comprehensively profile mutations in hematologic malignancies, we developed a targeted multimodal NGS assay that can detect SNVs, InDels, fusions, gene expression, and copy number variations from total nucleic acid (TNA) in a single tube workflow. Methods: TNA was extracted from peripheral blood and bone marrow specimens from patients with hematologic cancers. TNA was used to prepare libraries then sequenced on a NovaSeq 6000. DNA variants were compared to results from DNA NGS assays and RNA fusions were compared to FISH and RT-PCR. Results: Our multimodal NGS assay can efficiently use TNA to detect mutations simultaneously within the DNA and RNA in a single tube workflow. From 100 fusion positive samples, we detected fusions in all samples and >25 different fusions were detected. Our NGS assay was 100% concordant with the BCR-ABL1 qRT-PCR assay in samples with an IS value of >0.5, 92.7% concordant with the ArcherDX Heme NGS assay, and 100% sensitive in detecting high-confidence fusions. In 5 previously tested BCR-ABL1 positive samples, we confirmed the RNA expression as well as detected pathogenic DNA variants, including JAK2 p.V617F, U2AF1 p.S34F, ASXL1 p.E635Rfs*15, BRCA p.S1982Rfs*22, and DNMT3A p.S708Vfs*71. In another patient, we found multiple pathogenic mutations (ASXL1 and JAK2), in addition to a BCR-FGFR1 fusion. Two PML(e4)-RARA and PML(e6)-RARA isoforms were detected and confirmed in one sample, illustrating the high resolution that could be used to help monitor the patient. Three fusions involving CXCR4 (CXCR4-FOSL2, CXCR4-DDX5, and ARID5A-CXCR4), a receptor known to promote proliferation, migration and resistance to chemotherapy were also detected in addition to CXCR4 over-expression in three patients. In another patient, we confirmed a KMKT2A-ARHGEF12/del(11)(q23q23) aberration by NGS that was missed by cytogenetics. In one sample, we confirmed expression of 3 out of 4 different MYC fusions (MYC-BCL6, MYC-IgH, IgH-MYC); only NGS could identify the fusion orientation, illustrating the high resolution of NGS over FISH. In several patients with IgH-BCL1 translocations, we expected BCL1 overexpression. Although DNA PCR results were mostly negative, we discovered increased expression in a subset of these samples. This suggests that despite the detection of the fusion by FISH, a subset may lack gene expression which may suggest a different biological or clinical significance. Conclusion: Our findings demonstrate the value that a comprehensive profile provides to diagnostic tests. NGS has high resolution and by targeting RNA we can detect more fusions than traditional DNA approaches. Comparison of FISH and NGS results show that detecting a functional mutation maybe important for characterizing a disease. However, both may need to be combined for a complete picture to improve detection and characterization of various hematologic diseases. Citation Format: Cynthie Wong, Brad Thomas, Yanglong Mou, Christophe Magnan, Segun Jung, Tibor Gyuris, Francys Alarcon, Eve Shinbrot, Fei Ye, Ryan Bender, Sally Agersborg, Lawrence Weiss, Vincent Funari. A comprehensive genomic profiling approach to detect functional translocations and genomic alterations in a single tube workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2208.

Next-generation sequencing (NGS) provides a powerful high-throughput approach to identify and track B-cell immunoglobulin (IG) heavy chain clonality and to assess somatic hypermutation status in a massively parallel manner. The NGS-based clonality/SHM testing demonstrated superior performance over the conventional capillary electrophoresis (CE) methods in characterization of B-cell neoplasms. However, its broad utilization in clinical diagnostics requires extensive validation of assays as well as standardization of data interpretation. We present here an NGS assay designed and validated specifically for IG clonality/SHM characterization of B-cell malignancies. The assay was designed to simultaneously target the Leader, FR1, FR2, and FR3 regions of the IGH gene to identify clonal IGH VH-JH rearrangement as well as the IGK gene to identify clonal IGK VK-JK, VK-Kde, and INTR-Kde rearrangement. The validation was performed to assess assay accuracy, specificity, sensitivity, repeatability, and reproducibility, with both pre-characterized reference controls and prospectively collected clinic peripheral blood and bone marrow aspirate specimen included. Triplicates were included and testing was performed at different times and by different operators to assess the assay precision. Over 60 clinic peripheral blood and bone marrow aspirate specimens were collected, including patients with B-cell malignancies such as CLL, B-ALL, and DLBCL as well as heathy donors, processed within 7 days for DNA extraction, and assessed by the NGS assay for IGH clonality and SHM assessment. Assay sensitivity as low as 2.5% for baseline clonality and as low as 0.001% for tracking MRD were observed. Near-perfect assay specificity and precision were observed at these sensitivity levels. The validated NGS assay was further qualified by ERIC (the European Research Initiative on CLL) with a certificate granted to standardize the data interpretation of this assay for testing in chronic lymphocytic leukemia. In conclusion, a NGS IG clonality/SHM assay was analytically and clinically validated under medical oversight, with a demonstrated rigor of the test by its high sensitivity/specificity and robust reproducibility. The clinical diagnostic testing results with this assay is interpreted in accordance with ERIC standards for reliable clinical reporting. Citation Format: Xin-Xing Tan, Tricia Peters, Meredith Berry, Kristyn Jeter, Brigitte Lovell, Victor Venegas, Yanglong Mou, Brad Thomas, Vincent Funari, Josette William. Validation of a next generation sequencing clinical assay for detection of immunoglobulin heavy chain clonality and somatic hypermutation in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2778.

Background: Many new guidelines require a comprehensive genomic profiling approach for diagnosis, risk stratification and therapy decisions. Limitations in sample quantity and throughput may limit the number of single biomarker tests (FISH, karyotyping, sequencing, qRT-PCR, etc.) that can be performed for the patient. There are currently multiple commercial NGS assay options for total nucleic acid, however they involve independent parallel workflows and twice the amount of sample and effort. Here we developed a novel consolidated DNA/RNA workflow in a single-tube assay utilizing custom QIAseq multimodal chemistry. This simplified workflow enables a discovery approach of all critical DNA/RNA abnormalities in hematologic malignancies, extending our NGS capabilities to large structural changes, RNA fusions and expression. Methods: 297 Heme-focused genes and 14 chromosomes were targeted in the genome, along with 213 RNA genes targeting 712 exons involved in known fusions in the transcriptome using a custom QIAseq workflow. Captured DNA/RNA targets from 135 patients were sequenced with unique dual indices on an Illumina's NovaSeq 6000. Coverage and variant allele frequency from all gene and chromosomal targets in 25 disease free patients was compared to the same genomic targets in 76 patients that were referred for a suspected hematological malignancy (e.g. MDS, CML, AML, ALL, etc.). We compared results from our custom algorithm to karyotyping and FISH. In addition, we assessed the relationship between structural changes and the average mutation load for each indication. Positives gene fusions were confirmed by qRT-PCR or Sanger sequencing. Results: Cytogenetic abnormalities in 30/32 patients were confirmed by karyotyping and FISH; two cases with abnormalities were missed by NGS. NGS detected additional abnormalities not detected by cytogenetics, including a case of loss of chr17 including deletion of driver genes, NF1 and SUZ12. No significant relationship between chromosome abnormalities and tumor mutation burden was observed. However, patients referred for myeloid disorders with structural abnormalities had a significantly higher mutational burden (p<9.12x10−7). Mutational load in these patients was significantly associated with chromosome 17 abnormalities, primarily loss (p<0.013). qRT-PCR confirmed 100% of BCR-ABL fusions (p210, p190) in all patients (59/59) with International Scale (IS) percentages ranging from 2.4-100%. Notably, we confirmed two clinically significant fusions, SET-NUP214 and RUNX1-RUNX1T1 in two patients not previously interrogated by qRT-PCR. The SET-NUP214 fusion is normally associated with acute lymphoblastic leukemia (ALL) was identified in a patient with suspected CML. We also confirmed PCALM-MLLT10 gene fusion was detected in a commercial universal human RNA reference material, commonly used in expression profiling studies. Conclusions: This study confirms the validity and utility of simple but efficient comprehensive genomic profiling for use in hematologic malignancies. Coupled with FISH and cytogenetics tests, NGS can offer a better diagnostic and prognostic testing service for patients with hematologic disease to assist in treatment selection as well as precise patient care. Citation Format: Segun C. Jung, YongXin Yu, Yanglong Mou, Hyunjun Nam, Cynthie Wong, Samuel Koo, Brad Thomas, Forrest Blocker, Derek Lyle, Ryan Bender, Sally Agersborg, Lawrence M. Weiss, Vincent A. Funari. A comprehensive genomic profiling approach to interrogate hematologic malignancies using a novel multimodal next generation sequencing assay in a single-tube [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 182.

Background: A cost effective and comprehensive genomic profiling (CGP) approach for diagnosis, risk stratification and therapy would be useful for the evaluation of oncologic specimens. Available approaches involving additive testing for DNA and RNA abnormalities through traditional methods (e.g. Sanger, FISH, cytogenetics, qRT-PCR) are not comprehensive, require multiple different workflows and are sample consuming, often resulting in incomplete testing. While there are next generation sequencing (NGS) assays designed for detecting DNA and RNA abnormalities, they have separate workflows that require twice the amount of sample and effort. To address this, we developed a novel total nucleic acid (TNA) extraction method and single tube workflow utilizing TNA and a custom multimodal chemistry designed for hematologic malignancies. This consolidated workflow enables an efficient discovery based approach for both DNA/RNA abnormalities including single nucleotide variants (SNVs), InDels, copy number variants (CNVs), large structural changes from DNA and gene fusions and gene expression levels from RNA. This method maximizes data derived from valuable samples while delivering a comprehensive profile of the patient's tumor which can help guide therapeutic and clinical decisions. Methods: Total nucleic acid (TNA) was extracted from bone marrow and peripheral blood of 95 patients (CML, CMML, CLL, AML and myeloid disorders). 297 genes that have DNA mutations specific to hematological cancers were targeted, along with 213 genes that were targeted for clinically significant RNA abnormalities. Enriched genomic and transcriptomic regions of interest from 85 patients were successfully sequenced with unique dual indices on an Illumina NovaSeq 6000. DNA variant detection as well as fusion detection from RNA were compared to traditional orthogonal NGS assays that use DNA input or compared to qRT-PCR and Sanger sequencing assays that use RNA as input. Results: In this study, we developed an efficient and high-quality TNA extraction method that can purify enough total nucleic acid from bone marrow, peripheral blood, cytogenetic pellets, flow suspension, and FFPE samples for the downstream NGS assay. The average OD 260/280 value was 1.9 and the OD 260/230 was 2.18. After sequencing, 256/262 (97.7% accuracy) SNV and Indel variants that were candidate pathogenic mutations were concordant from 38 patients. Meanwhile, 100% (7/7) of all BCR/ABL1 gene fusions which had an international scale (IS) value above 6.4% were concordant. In addition, 69 fusion positive samples containing 20 unique gene fusions which had been previously reported by an independent ArcherDX assay designed specifically for gene fusions were also evaluated with this chemistry. Analysis revealed a 92.5% (64/69) concordance. More importantly, the QIAseq multimodal TNA NGS assay detected both DNA and RNA abnormalities in a single tube. For example, in one myeloid leukemia patient, we not only identified pathogenic variants of ASXL1 and JAK2 which had been previously detected by a DNA NGS assay, but also detected a concurrent BCR-FGFR1 fusion which had been previously reported by a FISH assay. Moreover, we were able to provide more comprehensive genomic profiling by investigating many DNA and RNA abnormalities simultaneously. In our study, for 5 patients that previously been tested for BCR-ABL1 fusion only, we are able to assess BCR-ABL1 fusion status from RNA as well as identify pathogenic DNA variants at the same time, including JAK2 p.V617F, U2AF1 p.S34F, ASXL1 p.E635Rfs*15, BRCA p.S1982Rfs*22, and DNMT3A p.S708Vfs*71, which provides valuable information to assist diagnosis and treatment in a cost effective and efficient way. Conclusions: We developed a single tube TNA based workflow with a custom multimodal chemistry that simultaneously detects many DNA and RNA abnormalities in a cost effective and efficient way while reducing sample requirements. This unique TNA NGS assay provides comprehensive genomic profiling for hematologic malignancies and improves the diagnostic testing options for precise patient care. Disclosures Yu: NeoGenomics: Current Employment. Alarcon:NeoGenomics: Current Employment. Mou:NeoGenomics: Current Employment. Jung:NeoGenomics: Current Employment. Nam:NeoGenomics: Current Employment. Thomas:NeoGenomics: Current Employment. Keeler:NeoGenomics: Current Employment. Shinbrot:NeoGenomics: Current Employment. Magnan:NeoGenomics: Current Employment. Bender:NeoGenomics: Current Employment. Jiang:NeoGenomics: Current Employment. Agersborg:NeoGenomics: Current Employment. Weiss:Bayer: Other: speaker; Genentech: Other: Speaker; Merck: Other: Speaker; NeoGenomics: Current Employment. Ye:NeoGenomics: Current Employment. Funari:NeoGenomics: Current Employment.

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