PCR-based Enrichment Research Articles

Salmonella and other Enterobacteriaceae in conventional and organic vegetables grown in Brazilian farms.

This study aimed to assess the microbiological profile of conventional and organic vegetables grown in Brazilian farms through the detection of Salmonella and other Enterobacteriaceae. A total of 200 samples (100 conventional and 100 organic), including leafy greens, spices/herbs, and other unusual vegetables, were submitted to the enumeration of Enterobacteriaceae by plating on VRBG agar. Moreover, colonies of Enterobacteriaceae were randomly selected and submitted to identification by MALDI-TOF MS. Samples were also tested for Salmonella, using culture-based and PCR-based enrichment methods. The mean counts of Enterobacteriaceae in conventional and organic vegetables were 5.1 ± 1.5 and 5.4 ± 1.4 log CFU/g, respectively (P > 0.05). A total of 18 genera (including 38 species) of Enterobacteriaceae were identified, and the most frequent ones found in samples from both farming systems were Enterobacter (76%) and Pantoea (68%). Salmonella was identified in 17 samples (8.5%): nine (4.5%) in conventional and eight (4.0%) in organic vegetables. These results indicate that the farming system had no impact on the Enterobacteriaceae populations and rates of Salmonella and revealed unsatisfactory microbiological safety of some samples, mainly due to the presence of Salmonella. These findings highlight the need for control measures during vegetable production, regardless of the farming system, to reduce microbial contamination and the risks of foodborne illnesses.

Targeted High-Throughput Sequencing Enables the Detection of Single Nucleotide Variations in CRISPR/Cas9 Gene-Edited Organisms.

Similar to genetically modified organisms (GMOs) produced by classical genetic engineering, gene-edited (GE) organisms and their derived food/feed products commercialized on the European Union market fall within the scope of European Union Directive 2001/18/EC. Consequently, their control in the food/feed chain by GMO enforcement laboratories is required by the competent authorities to guarantee food/feed safety and traceability (2003/1829/EC; 2003/1830/EC). However, their detection is potentially challenging at both the analytical and interpretation levels since this requires methodological approaches that can target and detect a specific single nucleotide variation (SNV) introduced into a GE organism. In this study, we propose a targeted high-throughput sequencing approach, including (i) a prior PCR-based enrichment step to amplify regions of interest, (ii) a sequencing step, and (iii) a data analysis methodology to identify SNVs of interest. To investigate if the performance of this targeted high-throughput sequencing approach is compatible with the performance criteria used in the GMO detection field, several samples containing different percentages of a GE rice line carrying a single adenosine insertion in OsMADS26 were prepared and analyzed. The SNV of interest in samples containing the GE rice line could successfully be detected, both at high and low percentages. No impact related to food processing or to the presence of other crop species was observed. The present proof-of-concept study has allowed us to deliver the first experimental-based evidence indicating that the proposed targeted high-throughput sequencing approach may constitute, in the future, a specific and sensitive tool to support the safety and traceability of the food/feed chain regarding GE plants carrying SNVs.

RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting variants of concern (VOC) while also providing virus quantitation data. However, detection sensitivities of both RT-qPCR and sequencing can be impacted through losses occurring during sample handling, virus concentration, nucleic acid extraction, and RT-qPCR. Therefore, process limit of detection (PLOD) assessments are required to estimate the gene copies of target molecule to attain specific probability of detection. In this study, we compare the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab) for detection of SARS-CoV-2 to that of ATOPlex sequencing by seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in greater detection rates than individual assays, suggesting that application of multiple assays is better suited for the trace detection of SARS-CoV-2 from wastewater samples. Furthermore, while ATOPlex offers a promising approach to SARS-CoV-2 wastewater surveillance, this approach appears to be less sensitive compared to RT-qPCR under the experimental conditions of this study, and may require further refinements. Nonetheless, the combination of RT-qPCR and ATOPlex may be a powerful tool to simultaneously detect/quantify SARS-CoV-2 RNA and monitor emerging VOC in wastewater samples.

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.

As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

Target region sequencing and applications in plants

Fine mapping quantitative trait loci (QTL) and understanding the molecular mechanism and underlying functions of genes are vital to the advancement of plant research and plant breeding. Target region sequencing (TRS) provides an efficient way to assist in accomplishing these goals. Once a genomic region is found to be responsible for a phenotype, TRS can be deployed to characterize that region. Understanding the available methodologies to perform TRS will be advantageous when determining how to proceed with specific projects. In this article, we reviewed and summarized four main methods that are being used for TRS in plants, including hybrid capture, PCR-based enrichment, DNA fragment circularization, and RNA capture. We also discussed the potential uses of these technologies in plant research, especially the marker discovery for breeding selection. The TRS techniques outlined in this review have the potential to become commonplace in plant research, thereby having a large impact.

Targeted Next-Generation Sequencing of 117 Routine Clinical Samples Provides Further Insights into the Molecular Landscape of Uveal Melanoma.

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1 and EIF1AX, most being linked to metastatic-risk. To gain further insight into the molecular landscape of UM, we designed a targeted next-generation sequencing (NGS) panel to detect SCNA and mutations in routine clinical UM samples. We compared hybrid-capture and amplicon-based target enrichment methods and tested a larger cohort of primary UM samples on the best performing panel. UM clinical samples processed either as fresh-frozen, formalin-fixed paraffin embedded (FFPE), small intraocular biopsies or following irradiation were successfully profiled using NGS, with hybrid capture outperforming the PCR-based enrichment methodology. We identified monosomy 3 (M3)-UM that were wild-type for BAP1 but harbored SF3B1 mutations, novel frameshift deletions in SF3B1 and EIF1AX, as well as a PLCB4 mutation outside of the hotspot on exon 20 coinciding with a GNAQ mutation in some UM. We observed samples that harboured mutations in both BAP1 and SF3B1, and SF3B1 and EIF1AX, respectively. Novel mutations were also identified in TTC28, KTN1, CSMD1 and TP53BP1. NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. BAP1 and SF3B1 mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome.

Optimized PCR-Based Enrichment Improves Coverage Uniformity and Mutation Detection in Mitochondrial DNA Next-Generation Sequencing

Next-generation sequencing-based methods have been commonly used for detecting mutations of mitochondrial genome (mtDNA). PCR amplification is a highly effective method of mtDNA enrichment before sequencing. However, it has been observed that highly variable sequencing depth within PCR amplicons severely reduces the coverage uniformity and accuracy of mutation calling. Therefore, it is necessary to develop an optimized PCR-based strategy for mtDNA sequencing. Herein, the effect of DNA quality on the efficiency of PCR amplification was analyzed and the effects of different primer-design methods, including the number of primer pairs, overlap length of amplicons, and modification of primers, on coverage uniformity and mutation calling in mtDNA sequencing were assessed. Results showed that DNA quality significantly affected the efficiency of PCR amplification. Importantly, over- and under-representation of coverage depth at overlap regions of amplicons were observed when amplicons were not modified and overlap was shorter than two sequencing fragment sizes (800 bp). Then, under-representation was overcome by increasing the overlap length of the amplicons, and over-representation was effectively reduced by 5'-block modification of primers and sticky-end ligation of amplicons. Moreover, findings indicated that these two optimized PCR-based sequencing strategies effectively improved mutation calling in primer-binding regions. Optimized PCR-based mtDNA enrichment and sequencing approaches have been established, which laid a foundation for accurate mutation detection of mtDNA in diseases.

Phylogeny of the Australian Solanum dioicum group using seven nuclear genes, with consideration of Symon's fruit and seed dispersal hypotheses.

The dioecious and andromonoecious Solanum taxa (the “S. dioicum group”) of the Australian Monsoon Tropics have been the subject of phylogenetic and taxonomic study for decades, yet much of their basic biology is still unknown. This is especially true for plant-animal interactions, including the influence of fruit form and calyx morphology on seed dispersal. We combine field/greenhouse observations and specimen-based study with phylogenetic analysis of seven nuclear regions obtained via a microfluidic PCR-based enrichment strategy and high-throughput sequencing, and present the first species-tree hypothesis for the S. dioicum group. Our results suggest that epizoochorous trample burr seed dispersal (strongly linked to calyx accrescence) is far more common among Australian Solanum than previously thought and support the hypothesis that the combination of large fleshy fruits and endozoochorous dispersal represents a reversal in this study group. The general lack of direct evidence related to biotic dispersal (epizoochorous or endozoochorous) may be a function of declines and/or extinctions of vertebrate dispersers. Because of this, some taxa might now rely on secondary dispersal mechanisms (e.g. shakers, tumbleweeds, rafting) as a means to maintain current populations and establish new ones.

Combined targeted DNA and RNA sequencing of advanced NSCLC in routine molecular diagnostics: Analysis of the first 3,000 Heidelberg cases.

Tyrosine kinase inhibitors currently confer the greatest survival gain for nonsmall cell lung cancer (NSCLC) patients with actionable genetic alterations. Simultaneously, the increasing number of targets and compounds poses the challenge of reliable, broad and timely molecular assays for the identification of patients likely to benefit from novel treatments. Here, we demonstrate the feasibility and clinical utility of comprehensive, NGS-based genetic profiling for routine workup of advanced NSCLC based on the first 3,000 patients analyzed in our department. Following automated extraction of DNA and RNA from formalin-fixed, paraffin-embedded tissue samples, parallel sequencing of DNA and RNA for detection of mutations and gene fusions, respectively, was performed using PCR-based enrichment with an ion semiconductor sequencing platform. Overall, 807 patients (27%) were eligible for currently approved, EGFR-/BRAF-/ALK- and ROS1-directed therapies, while 218 additional cases (7%) with MET, ERBB2 (HER2) and RET alterations could potentially benefit from experimental targeted compounds. In addition, routine capturing of comutations, e.g. TP53 (55%), KEAP1 (11%) and STK11 (11%), as well as the precise typing of fusion partners and involved exons in case of actionable translocations including ALK and ROS1, are prognostic and predictive tools currently gaining importance for further refinement of therapeutic and surveillance strategies. The reliability, low dropout rates (<5%), minimal tissue requirements, fast turnaround times (6 days on average) and lower costs of the diagnostic approach presented here compared to sequential single-gene testing, highlight its practicability in order to support individualized decisions in routine patient care, enrollment in molecularly stratified clinical trials, as well as translational research.

Rapid whole-genome based typing and surveillance of avipoxviruses using nanopore sequencing

Avian pox is an infectious disease caused by avipoxviruses (APV), resulting in cutaneous and/or tracheal lesions. Poxviruses share large genome sizes (from 130 to 360 kb), featuring repetitions, deletions or insertions as a result of a long-term recombination history.The increasing performances of next-generation sequencing (NGS) opened new opportunities for surveillance of poxviruses, based on timely and affordable workflows. We investigated the application of the 3rd generation Oxford Nanopore Minion technology to achieve real-time whole-genome sequencing directly from lesions, without any enrichment or isolation step.Fowlpox lesions were sampled on hens, total DNA was extracted and processed for sequencing on a MinION, Oxford Nanopore. We readily generated whole APV genomes from cutaneous or tracheal lesions, without any isolation or PCR-based enrichment: Fowlpox virus reads loads ranged from 0.75% to 2.62% and reads up to 61 kbp were generated and readily assembled into 3 APV complete genomes. This long read size eases the assembly step and lowers the bioinformatics capacity requirements and processing time compared to huge sets of short reads. The complete genome analysis confirmed that these Fowlpox viruses cluster within clade A1 and host full length reticuloendotheliovirus (REV) inserts. The pathobiological relevance of REV insert, although a classical feature of fowlpoxviruses (FPVs), should be further investigated. Surveillance of emerging poxviruses could greatly benefit from real-time whole genome sequencing.

30. Clinical utility of NGS in the understanding of crosstalk between nuclear cancer genes and mitochondrial genome in tumorigenesis

Reprogramming of energy metabolism is one of the hallmarks of cancers. Dynamic bioenergetics changes can be evidenced by the detection of deleterious mutations in nuclear or mitochondrial genes related to energy metabolism and cancer during tumorigenesis. In order to identify these molecular changes, we developed a comprehensive next-generation sequencing (NGS) approach by target capture-sequencing of 183 cancer-related genes with an average read depth of approximately 1000X. The deep coverage facilitates simultaneous detection of exonic copy number variants (CNVs) and single nucleotide variants (SNVs) at low somatic mosaicism in one comprehensive assessment. Additionally, the circular mitochondrial genome is analyzed as a single amplicon using one pair of back to back primers for the long-range PCR-based enrichment coupled with massively parallel sequencing at an average coverage of ∼20,000X. Thirty Trio (tumor, adjacent tissue, and blood) of colon cancers (20 carcinomas and 10 polyps) were studied. Variants detected are classified according to ACMG guidelines. Out of 30 Trio samples, germline variants were found in 11 patients, 4 of which have pathogenic mutations. Somatic nuclear driver mutations are present in all tumors at >15%. As for mtDNA variants, 1 out of 30 carries deleterious germline mtDNA mutations and 5 harbor somatic pathogenic mtDNA variants. Upon completion of data analysis of 30 Trio samples, it demonstrates energy reprogramming is the pathway intimately correlated with tumorigenesis even though every tumor develops its unique mechanism leading to tumorigenesis.

Abstract 4651: Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit

Abstract Introduction: Next-generation sequencing (NGS) has been being rapidly adopted in clinical research to align actionable variants in tumors to targeted therapies or clinical trials. Comprehensive NGS-based tumor profiling assays are an efficient and effective method to characterize a variety of clinically relevant somatic mutations. We have developed a targeted NGS cancer gene panel that focuses on actionable gene mutations and employs a NGS library preparation method based on multiplex PCR enrichment in an integrated fluidic circuit (IFC) and the automated JunoTM System. Methods: 53 actionable cancer genes were selected based on both clinical and research knowledge that covers 15 solid tumor types. Assays for the selected regions of the 53 genes were designed by an internal assay design pipeline. Multiplex PCR using target-specific primers is conducted in an LP—48.48 IFC on the Fluidigm Juno™ system, where up to 48 DNA samples can be processed simultaneously. A unique barcode is incorporated in one of the PCR primers to distinguish individual samples. To evaluate the assay performance, three sets of cell line gDNA samples were identified from commercial sources: set 1: 22 commercial reference samples, set 2: 12 samples for single nucleotide variants (SNVs) and indels, and set 3: 12 samples for copy number variations (CNVs). Each sample was tested on the IFC in 4 replicates. PCR products harvested from the IFC were pooled and purified with Agencourt® AMPure® XP magnetic beads. A second PCR step using a universal primer pair was performed to add sequencing adapters. After a second purification, the DNA library was sequenced on a NextSeq™ 500 system. The data analysis was performed by a service provider partner (GenomOncology). Results: A total of 1,508 primer pairs with an average insert size of 155 bp were selected to cover SNVs, indels, and CNVs. The assays were separated into 44 pools to minimize the interaction between assay primers and improve performance. The percentage of reads mapped to the genome was 98.9%, and the percentage of reads that mapped to amplicons was 96.8%. Mutation detection sensitivity was 4% variant allele frequency (VAF.) In 47 selected samples, the total SNVs, CNVs and indels represented are 182, 154 and 28, respectively. For SNVs, the positive percent agreement (PPA) is 1.0 and positive predictive value (PPV) is 0.974. For CNVs, the PPA is 1.0 and PPV is 0.969 and for indels, the PPA and PPV are 1.0 and 0.966, respectively. Overall concordance is 0.99. Conclusions: A targeted NGS cancer panel employing PCR-based enrichment on an IFC has been developed and yielded high quality of libraries generated on the JunoTM system for detecting SNVs, indels and CNV in solid tumor samples. This panel covers actionable targets in 53 cancer genes and utilizes a microfluidic device to provide a simple streamlined workflow for library preparation of up to 48 DNA samples per IFC. Citation Format: Peilin Chen, Jaibiao Gong, David Wang, Devin Do, Lianne McLean, Tom Goralski. Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit [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 4651.

344 Detection of ERG Rearrangement in Formalin-Fixed Paraffin-Embedded Tissue of Prostatic Adenocarcinoma by Targeted RNA-seq

Chromosomal translocations involving the erythroblast transformation-specific (ETS) transcription factors, such as ERG and ETV1, are frequent events in human prostate cancer pathogenesis. In particular, the TMPRSS2-ERG fusion gene has recently been found to be the most frequent gene rearrangement in prostate cancers. Previously, IHC stain and FISH assay have been used to detect ERG translocation. However, these methods do not provide detailed information, such as the exact breakpoint and partner gene. Here, we want to test the feasibility of detection of ERG translocation by targeted RNA-seq. Tissue microarray (TMA) was constructed from 27 cases of prostatic adenocarcinoma. TMA slides were subjected to ERG immunostaining. Cases with positive ERG staining were identified. For targeted RNA-seq, we used Archer FusionPlex (ArcherDX, Boulder, CO) Solid Tumor Kit, which uses Anchored Multiplex PCR-based enrichment method and can detect fusions associated with >50 genes. Both ERG-positive and -negative tissue was subject for this assay. Eight samples (four of ERG+ prostate cancer and four of ERG– tissue) generated a total of two GB sequencing data. ERG translocation was not found in any of the four samples of ERG– tissue. TMPRSS2-ERG fusion was identified in one of the samples of ERG+ prostate cancer. Our preliminary test demonstrated that it is feasible to detect ERG rearrangement in formalin-fixed paraffin-embedded tissue by targeted RNA-seq, which could provide much more detailed information than other traditional methods. However, this test is still very challenging and requires strong bioinformatics support.

PyAmpli: an amplicon-based variant filter pipeline for targeted resequencing data

BackgroundHaloplex targeted resequencing is a popular method to analyze both germline and somatic variants in gene panels. However, involved wet-lab procedures may introduce false positives that need to be considered in subsequent data-analysis. No variant filtering rationale addressing amplicon enrichment related systematic errors, in the form of an all-in-one package, exists to our knowledge.ResultsWe present pyAmpli, a platform independent parallelized Python package that implements an amplicon-based germline and somatic variant filtering strategy for Haloplex data. pyAmpli can filter variants for systematic errors by user pre-defined criteria. We show that pyAmpli significantly increases specificity, without reducing sensitivity, essential for reporting true positive clinical relevant mutations in gene panel data.ConclusionspyAmpli is an easy-to-use software tool which increases the true positive variant call rate in targeted resequencing data. It specifically reduces errors related to PCR-based enrichment of targeted regions.

High-throughput PCR assay design for targeted resequencing using primerXL

BackgroundAlthough the sequencing landscape is rapidly evolving and sequencing costs are continuously decreasing, whole genome sequencing is still too expensive for use on a routine basis. Targeted resequencing of only the regions of interest decreases both costs and the complexity of the downstream data-analysis. Various target enrichment strategies are available, but none of them obtain the degree of coverage uniformity, flexibility and specificity of PCR-based enrichment. On the other hand, the biggest limitation of target enrichment by PCR is the need to design large numbers of partially overlapping assays to cover the target.ResultsTo overcome the aforementioned hurdles, we have developed primerXL, a state-of-the-art PCR primer design pipeline for targeted resequencing. It uses an optimized design criteria relaxation cascade and a thorough downstream in silico evaluation process to generate high quality singleplex PCR assays, reducing the need for amplicon normalization, and outperforming other target enrichment strategies and similar primer design tools when considering assay quality, coverage uniformity and target coverage. Results of four different sequencing projects with 2348 amplicons in total covering 470 kb are presented. PrimerXL can be accessed at www.primerxl.org.ConclusionPrimerXL is an state-of-the-art, easy to use primer design webtool capable of generating high-quality targeted resequencing assays. The workflow is fully customizable to suit every researchers’ needs, while an innovative relaxation cascade ensures maximal target coverage.

Abstract P2-03-10: A fit-for-purpose NGS system that reports ERBB2 (HER2) mutations and copy number variants for clinical trials research and drug development

Abstract Introduction: HER2-positive breast cancers comprise 20-30% by subtype and represent the second poorest prognosis. In addition to HER2 signaling activation via protein overexpression or gene amplification (measured by IHC or ISH), somatic mutations in the ERBB2 (HER2) gene have been identified in 2-4% of breast cancers and reported to drive HER2 pathway signaling and tumorigenesis. Irreversible HER2 tyrosine kinase inhibitors such as neratinib have been shown to potently inhibit breast tumors harboring somatic HER2 activating mutations and are under active clinical investigation. A targeted molecular characterization strategy is thus needed to support clinical trial research. Here, we present an integrated NGS system that identifies multiple classes of ERBB2aberrations, including single nucleotide variants, insertion/deletion mutations, and copy number variations (CNVs). Methods: Genomic DNA was isolated from FFPE human tumor tissues or FFPE-embedded engineered cell lines expressing ERBB2 mutations. Pre-analytical sample QC was performed by a novel qPCR assay that quantifies amplifiable DNA templates suitable for library preparation. NGS libraries were prepared using the QuantideX® NGS ERBB2 Mutations assay, with two-pool PCR-based enrichment to detect multiple types of ERBB2 genomic alterations. Libraries were sequenced (Illumina MiSeq®) and analyzed using QuantideX® Reporter software, directly incorporating the pre-analytical QC data from each DNA sample to improve variant call accuracy. Results: The QuantideX® NGS ERBB2 Mutations system targets 28% of ERBB2 coding bases covering exons 8 and 17 - 24, including codons 713-989 of the tyrosine kinase domain. The assay also detects focal, whole-arm and polysomy-induced HER2 amplification using sentinel flanking amplicons, and amplicons covering CEN17 and copy number-neutral chromosomal regions identified in the TCGA breast cancer cohort. The assay was analytically validated in a CLIA-laboratory which included an analysis of 6 mixtures and titrations of synthesized DNA bearing 31 ERBB2 mutations, 17 engineered cell lines and FFPE cell-line blocks, and 12 surgically-resected FFPE carcinomas. A multi-day/operator study revealed 100% sensitivity and PPV for detection of ERBB2 SNVs, indels and complex variants down to 5% mutation positive in wild-type DNA. The NGS system was also used to profile ERBB2 variants in 473 FFPE human tumor tissues, including 280 breast carcinomas. Mutation-positive samples were confirmed by an orthogonal NGS method. A subset of 46 FFPE tissue samples were assessed for CNVs by NGS and compared with matched droplet digital PCR (ddPCR) analysis. A correlation of R2 &amp;gt; 0.91 between NGS and ddPCR underscores the potential of the assay to reveal both ERBB2 mutations and copy number changes. Conclusions: The QuantideX® NGS ERBB2system integrates optimized wet- and dry-bench elements to achieve broad and accurate detection of ERBB2 mutations and copy number variants from challenging FFPE specimens. The technology has the potential to advance breast cancer clinical research and aid in the selection of patients for clinical trials, particularly those that assess HER2-directed therapies such as afatinib, lapatinib, and neratinib. Citation Format: Zhu H, Zeigler R, Bridger S, Larson JL, Kincaid A, Cardwell R, Haynes BC, Andruss BF, Latham GJ. A fit-for-purpose NGS system that reports ERBB2 (HER2) mutations and copy number variants for clinical trials research and drug development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-10.

Size-selective separation and overall-amplification of cell-free fetal DNA fragments using PCR-based enrichment

This study aimed to establish a method for the selective amplification of cell-free fetal DNA (cffDNA) in maternal plasma and preserve the integrity of DNA fragments during amplification, thereby providing a sufficient amount of cffDNA to meet the requirement of routine non-invasive prenatal testing. We amplified DNA molecules in a one-reaction system without considering their particular sequences and lengths (overall amplification) by using PCR-based enrichment. We then modified PCR conditions to verify the effect of denaturation temperature on DNA amplification on various lengths of DNA (selective overall amplification). Finally, we used an optimum temperature range to amplify cffDNA selectively. Amplification results were validated by electrophoresis and real-time quantitative PCR. Our PCR-based enrichment efficiently amplified all DNA fragments with differing lengths within a single reaction system, as well as preserving the integrity of the DNA fragments. cffDNA was significantly amplified along with the selective amplification of small fragment maternal plasma DNA in an appropriate range of denaturation temperatures. We have established a PCR-based method for the simultaneous enrichment and amplification of cffDNA in order to meet the requirements of high cffDNA quantity for routine non-invasive prenatal testing.

Identification of Multiple, Patient-Specific MLL Fusion Transcript Isoforms in Childhood Leukemia Using Anchored Multiplex PCR-Based Enrichment (AMP-E)

Chromosomal translocations involving 11q23, resulting in rearrangements of the mixed lineage leukemia gene (MLL, re-named KMT2A) are frequent events in childhood leukemia. MLL is highly promiscuous, with approximately 80 fusions now characterized. Although fluorescence in situ hybridization (FISH) has high specificity for detecting MLL-rearrangements (MLL-r), sensitivity is limited and the translocation partner gene (TPG) cannot always be identified. In contrast, long-distance inverse-PCR (LDI-PCR) permits sequence-specific characterization of MLL breakpoints and the resultant fusion gene, which can then be used for monitoring minimal residual disease (MRD). A limitation of LDI-PCR is the relatively large input of DNA (≈ 1μg) required, with a blast cell percentage of > 20-30% to achieve sufficient sensitivity. Next-generation sequencing (NGS) approaches such as RNAseq and whole-genome sequencing (WGS) have the potential to identify multiple gene fusions, however their ability to detect the full spectrum of MLL fusions is limited by coverage, read depth and thereby cost. Such limitations can potentially be overcome with targeted sequencing panels, although their performance against "gold standard" assays, such as LDI-PCR, is unknown. We therefore aimed to assess the ability of a novel, targeted NGS approach for characterizing patient-specific MLLgene rearrangements from low inputs of RNA.The Archer™ FusionPlex™ Heme and Myeloid panels utilize anchored multiplex PCR-based enrichment (AMP-E) to rapidly enrich a number of targets, including MLL, creating libraries for NGS. The NGS libraries are generated using rapid workflows and are compatible with nucleic acid inputs of ≈ 20-200ng. Briefly, double stranded cDNA is generated from patient RNA and subjected to end repair, adenylation and ligation with unique, half-functional adaptors. Following two rounds of nested PCR with primers attached to common sequencing adaptors, the resulting target amplicons become functional and ready for clonal amplification and sequencing. Using AMP-E, we tested 23 paediatric MLL-r samples (15 ALL, 8 AML) that had previously been analyzed by LDI-PCR and were known to harbor 8 different MLL fusions, including MLL-AFF1 (n = 8), -MLLT3 (5), -MLLT10 (3), -ELL (2), -DCP1A (1), -MLLT1 (1), - AFF3 (1), and -TNRC18 (1). A patient sample known to express BCR-ABL1 was used as a positive control and a cytogenetically normal AML sample in remission was used as a negative control in each panel. The median blast count for samples analyzed was 86.1% (range 25%-97%). On average, 100ng of RNA was used per sample, with RIN values ranging from 2.7 to 9.1. Libraries generated using either the Archer™ FusionPlex™ Heme or Myeloid kit were sequenced to sufficient read depths by Illumina MiSeq® and NextSeq®, respectively. Bioinformatic analyses were performed with the Archer™ Analysis 4.1 software. Results were then compared with fusions identified by LDI-PCR.There was high concordance between AMP-E and LDI-PCR, with all MLL fusion genes identified by LDI-PCR also detected by AMP-E. Of note, an ALL sample with t(11;19), unable to be characterized by LDI-PCR, was identified by AMP-E to express MLL-MLLT1. The control BCR-ABL1 fusion was identified in every run and there were no false-negative results. Furthermore, AMP-E identified multiple MLL-fusion transcripts in 56.5% of patients. Analysis of paired diagnosis-relapse samples from an AML patient with MLL-MLLT3demonstrated that the two discrete transcripts present at diagnosis persisted at relapse, with emergence of a third transcript.In summary, detection of MLL gene fusions in acute leukemia using AMP-E is both sensitive and specific. The low RNA requirement, rapid workflow, compatibility with Illumina MiSeq® and cloud-based proprietary analysis software, together with the array of additional fusions and mutations detected by the Archer™ panels, show promise for translation into clinical diagnostic settings. The persistence of discrete transcript isoforms at relapse also highlights the potential for AMP-E to identify multiple, patient-specific MLL fusion transcripts which may have utility in refining prognostication, MRD monitoring and informing future functional studies of MLL-driven leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

A statistical approach to detection of copy number variations in PCR-enriched targeted sequencing data.

BackgroundMultiplex polymerase chain reaction (PCR) is a common enrichment technique for targeted massive parallel sequencing (MPS) protocols. MPS is widely used in biomedical research and clinical diagnostics as the fast and accurate tool for the detection of short genetic variations. However, identification of larger variations such as structure variants and copy number variations (CNV) is still being a challenge for targeted MPS. Some approaches and tools for structural variants detection were proposed, but they have limitations and often require datasets of certain type, size and expected number of amplicons affected by CNVs. In the paper, we describe novel algorithm for high-resolution germinal CNV detection in the PCR-enriched targeted sequencing data and present accompanying tool.ResultsWe have developed a machine learning algorithm for the detection of large duplications and deletions in the targeted sequencing data generated with PCR-based enrichment step. We have performed verification studies and established the algorithm’s sensitivity and specificity. We have compared developed tool with other available methods applicable for the described data and revealed its higher performance.ConclusionWe showed that our method has high specificity and sensitivity for high-resolution copy number detection in targeted sequencing data using large cohort of samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1272-6) contains supplementary material, which is available to authorized users.

Reliable Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue Using Single Molecule Tags

Sequencing of tumor DNA to detect genetic aberrations is becoming increasingly important, not only to refine cancer diagnoses but also to predict response to targeted treatments. Next-generation sequencing is widely adopted in diagnostics for the analyses of DNA extracted from routinely processed formalin-fixed, paraffin-embedded tissue, fine-needle aspirates, or cytologic smears. PCR-based enrichment strategies are usually required to obtain sufficient read depth for reliable detection of genetic aberrations. However, although the read depth relates to sensitivity and specificity, PCR duplicates generated during target enrichment may result in overestimation of library complexity, which may result in false-negative results. Here, we report the validation of a 23-gene panel covering 41 hotspot regions using single-molecule tagging of DNA molecules by single-molecule molecular inversion probes (smMIPs), allowing assessment of library complexity. The smMIP approach outperforms Sanger and Ampliseq-Personal Genome Machine-based sequencing in our clinical diagnostic setting. Furthermore, single-molecule tags allow consensus sequence read formation, allowing detection to 1% allele frequency and reliable exclusion of variants to 3%. The number of false-positive calls is also markedly reduced (>10-fold), and our panel design allows for distinction between true mutations and deamination artifacts. Not only is this technique superior, smMIP-based library preparation is also scalable, easy to automate, and flexible. We have thus implemented this approach for sequence analysis of clinical samples in our routine diagnostic workflow.