Target-specific Primers Research Articles

Highly Selective Multiplex Quantitative Polymerase Chain Reaction with a Nanomaterial Composite Hydrogel for Precise Diagnosis of Viral Infection

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.

CRISPR-Cas12a-Based Nucleic Acid Amplification-Free DNA Biosensor via Au Nanoparticle-Assisted Metal-Enhanced Fluorescence and Colorimetric Analysis

Cell-free DNA (cfDNA) has attracted significant attention due to its high potential to diagnose diseases, such as cancer. Still, its detection by amplification method has limitations because of false-positive signals and difficulty in designing target-specific primers. CRISPR-Cas-based fluorescent biosensors have been developed but also need the amplification step for the detection. In this study, for the first time CRISPR-Cas12a based nucleic acid amplification-free fluorescent biosensor was developed to detect cfDNA by a metal-enhanced fluorescence (MEF) using DNA-functionalized Au nanoparticle (AuNP). Upon activating the CRISPR-Cas12a complex by the target cfDNA and subsequent single-strand DNA (ssDNA) degradation between AuNP and fluorophore, MEF occurred with color changes from purple to red-purple. Using this system, breast cancer gene-1 (BRCA-1) can be detected with very high sensitivity in 30 min. This rapid and highly selective sensor can be applied to measure other nucleic acid biomarkers such as viral DNA in field-deployable and point-of-care testing (POCT) platform.

Universal Sensitive, Accurate and Precise Microchimerism Surveillance Solution for Allogeneic Hematopoietic Cell Transplant

The success of hematopoietic cell transplantation (HCT) is limited by a high rate of disease relapse. Post-HCT chimerism analysis is typically used to evaluate donor cell engraftment and success of therapy in allogeneic HCT. The detection of microchimerism, especially in the early period after allogeneic HCT, is an important high-risk factor for disease relapse (Ahci et al, 2017). However, common approaches based on semi-quantitative fluorescent PCR of short tandem repeats (STRs) suffer from low sensitivity and high result variability and often fail to detect microchimerism and predict patient outcomes (Schuurhuis et al, 2018). Here, we describe development and characterization of AlloHemeTM, a novel sensitive and precise chimerism surveillance solution with high accuracy for detection and quantification of low levels of patient cells which ultimately can lead to clinical disease recurrence. The method is compatible with chimerism assessment in whole blood, bone marrow, or specific cell subtypes. CD3, CD15, CD33 and CD34 markers are used to separate T cells, myeloid progenitors and granulocytes from blood and hematopoietic stem cells from bone marrow, respectively. A minimum of 8ng genomic DNA is used to amplify hundreds of single nucleotide polymorphisms (SNPs) using target specific primers in a single multiplex reaction. Because there is no need to select genetic variants that differ for specific donor-patient pair, the method is universally applicable to any allogeneic HCT. Relative contribution of SNP alleles is measured by Next Generation Sequencing (NGS) and % chimerism of post-HCT samples is calculated using an analysis pipeline incorporating open source and custom bioinformatics tools. To evaluate reproducibility of cell subtype separation, blood from 3 healthy volunteers was subjected to cell separation for CD3, CD15 and CD33 cells at four different timepoints after draw (24, 48, 72 and 96 hours). Flow cytometry analysis using non-competing antibodies targeting same cell populations demonstrated that the cell subtype isolation step consistently yields sub-populations with an average purity of 96% across all cell subtypes and timepoints evaluated (93-99% range). To evaluate assay sensitivity, accuracy and precision, genomic DNA samples (gDNA, Coriell Institute) were used to prepare 2 panels, A and B, each containing different mixtures of gDNAs to simulate post-HCT samples with varying contribution of patient's cells. Panel A contained mixtures of gDNA samples from unrelated individuals ranging from 0.01-50% and panel B from related (parent-child) ranging from 0.01-75%. Each mixture was tested in 6 (Panel A) or 5 (Panel B) independent replicates. The results show that the assay is accurate from 0.03% through 50% for panel A and 0.1% through 75% for panel B with minimal proportional and systematic bias (Figure 1). The assay precision is high, with a mean coefficient of variation (CV) of 2.6% for panel A and 6.1% for panel B across the entire linear range. For sample mixtures with chimerism levels greater than 1%, mean CV was 0.76% for panel A and 1.62% for panel B. AlloHeme results show that the test can detect microchimerism down to 0.03%. High result reproducibility is achieved through the use of a proprietary NGS process and automated data analysis and workflow, generating results in the CLIA lab with a turnaround time of 3 days. The test was developed with rigorous QC steps to ensure high purity and accuracy for each cell population analyzed, increasing confidence and sensitivity of results. Given previously published data on clinical value of microchimerism, AlloHeme has the potential for early relapse detection post-HCT. Future studies will evaluate the utility of AlloHeme in clinical setting with the goal to improve patient outcomes and survival rates. In summary, we have developed a sensitive, accurate and precise method for the universal detection and quantification of chimerism and potential early disease recurrence detection for allogeneic HCT patient surveillance. Because the method provides high sensitivity and consistent results for microchimerism detection, it may be particularly suited to help identify recurrence of primary disease in hematologic cancer patients with or without known cancer-associated genetic determinants. Disclosures Egidio: CareDx: Current Employment, Current equity holder in publicly-traded company. Janakiraman:CareDx: Current Employment. Mao:CareDx: Current Employment. Gulbahce:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Wong:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Marchis:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Ghosh:CareDx: Current Employment. Grskovic:CareDx: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.

Abstract 4255: Reliable identification of both known and novel gene fusions using a high-efficiency amplicon-based NGS panel

Abstract Gene fusions and RNA splicing errors are important events frequently involved in tumorigenesis, and often represent clinically addressable targets for various cancers. The reliable detection of such mutations via NGS requires the development of cutting-edge technologies capable of overcoming many challenges. These include detecting fusion mutations regardless of the complexity of fusion structures, removing the length and quantity constraints of FFPE RNA, reducing ribosomal RNA interference, eliminating false negatives and positives, revealing the existing copies of mutations, and improving the reproducibility and ease of workflow. Here we describe the development and validation of a fast and user-friendly RNA-based fusion detection technology - CleanPlex® Fusion. Adding a universal primer at the 3' end of single-stranded cDNA via template switching allows us to eliminate the constraint of template length, and hence amplify damaged RNA fragments, such as FFPE RNA. The presence of a target-specific primer on one side enables the detection of any mutations on the RNA fragments, including novel ones. With a panel of 61 primers (covering most of fusion mutations of NSCLC) annealing to the 3' end portion of RNA, we demonstrated the high on-target rate, mapping rate, and low rRNA rate and primer-dimer rate. We validated the amplification of each amplicon by using fragmented total RNA. Additionally, we also made a dual-primer multiplex PCR panel covering the same set of fusion mutations as defined by the single-primer panel, confirmed its coverage of all fusion mutations using a synthetic pool of fusion targets, and detected all commercial reference fusion mutations targeted by the panel. We again demonstrated the detection of such reference fusion mutations using the single-primer mPCR panel, and demonstrated that the single-primer method even detected fusion mutations outside the boundary of the dual primers. Moreover, we showed that the detected fusion reads significantly correlate with copy numbers determined by digital PCR. Lastly, we validated detection of fusion mutations in cell lines and FFPE RNA. CleanPlex® Fusion therefore represent a robust and efficient NGS method for detecting fusion mutations. Citation Format: Chenyu LI. Reliable identification of both known and novel gene fusions using a high-efficiency amplicon-based NGS panel [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 4255.

Stealth Adapted Viruses with Genetically Unstable Rhesus Monkey Cellular Sequences A Possible Forerunner of Complex Human Illnesses

Real time PCR is a widely used for molecular diagnosis of tuberculosis in routine diagnosis laboratory. There are many commercially avaliable system. The sensitivity of the system varies according to DNA extraction from the sample and target specific primers and probes and commercial systems. In this study, the sensitivity and specificity of AdvanSure TB/NTM real time PCR kit were evaluated for detecting mycobacteria in smear positive and negative sputum specimens of 35 active pulmonary tuberculosis patients. All specimens were corrected using EZN staining, solid (Löwenstein Jensen) and liquid culture (MGIT). Culture and clinical findings were used as gold standards in the evaluation of all PCR results. Overall true positivity was found 25 ( 71.42%). When smear negative samples were evaluated together with culture results, only 3 patients were found positive by PCR test in 10 samples (30%).

Evaluation of Advansure TB/NTM Real Time PCR Kit for Detection of Mycobacteria in Sputum Specimens

Real time PCR is a widely used for molecular diagnosis of tuberculosis in routine diagnosis laboratory. There are many commercially avaliable system. The sensitivity of the system varies according to DNA extraction from the sample and target specific primers and probes and commercial systems. In this study, the sensitivity and specificity of AdvanSure TB/NTM real time PCR kit were evaluated for detecting mycobacteria in smear positive and negative sputum specimens of 35 active pulmonary tuberculosis patients. All specimens were corrected using EZN staining, solid (Löwenstein Jensen) and liquid culture (MGIT). Culture and clinical findings were used as gold standards in the evaluation of all PCR results. Overall true positivity was found 25 ( 71.42%). When smear negative samples were evaluated together with culture results, only 3 patients were found positive by PCR test in 10 samples (30%).

In-particle stem-loop RT-qPCR for specific and multiplex microRNA profiling

Here we report a novel method of microRNA (miRNA) profiling with particle-based multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR). To achieve target-specific reaction in a particle, the stem-loop RT primer and forward primer for each target miRNA were chemically immobilized to the particle. Target-specific cDNA synthesis proceeds with the stem-loop RT primer and then qPCR subsequently proceeds with the forward primer to rapidly achieve a quantitative result. High-fidelity multiplex assay was also accomplished in a single PCR process by loading multiple particles for each specific miRNA. The method for primer supply in the particles, involving confinement of the target-specific RT and PCR primers in the matrix of particles, led to the reduction of nonspecific reactions and improved the selectivity of the miRNA assay while minimizing labor in a multiple target assay. Specifically, this particle-based assay enabled the differentiation of mature miRNA from precursor with selectivity of 270:1 in terms of amplification speed. This advanced method also showed good discrimination among highly homologous let-7 family members, with cross-reaction rates of less than 5%. We demonstrated a very simple process of five-plex miRNA profiling in total RNA, and the measured changes in expression level were consistent with those from a conventional singleplex method.

Prevalence and Diversity of Haplotypes of Sickle Cell Disease in the Eastern Province of Saudi Arabia

Hb F modulates sickle cell disease. Five major haplotypes of the β-globin gene cluster are associated with sickle cell disease. In the Eastern Province of Saudi Arabia, the Arab-Indian (AI) is most common. Single nucleotide polymorphism (SNP) genotyping (rs3834466, rs28440105, rs10128556, and rs968857) was carried out by nuclease allelic discrimination assay with target-specific forward and reverse primers, TaqMan probes, labeled with VIC and FAM. In 778 patients with sickle cell disease from the Eastern Province, a haplotype was assigned to 90.9% of all samples; 9.1% were classified as compound heterozygotes for the AI and an atypical haplotype. The distribution of haplotypes for 746 Hb S (HBB: c.20A > T) homozygotes was: 614 AI/AI, nine SEN/SEN (Senegal), 42 SEN/AI, nine CAM/CAM (Cameroon), one CAR (Central African Republic)/BEN (Benin), 71 AI/atypical. In Hb S/β-thalassemia (Hb S/β-thal), the distribution of Hb S haplotypes was: 22 AI/AI, one CAM/CAM, four AI/SEN, five AI/atypical. Mean Hb F in the haplotypes was: AI/AI 16.6 ± 7.5%, CAM/CAM 8.0 ± 4.1%, SEN/SEN 11.0 ± 5.1%, SEN/AI 15.1 ± 4.6%, AI/atypical 16.2 ± 6.5%. The presence of the SEN and CAM haplotypes was unexpected due to the apparent homogeneity of the population of the Eastern Province. We have successfully classified sickle cell disease haplotypes using the relatively inexpensive TaqMan assay for the first time. In addition, we have previously shown that children with AI haplotype have Hb F of 30.0% and mild disease, while in our cohort of adult AI patients, which might be the largest yet reported, Hb F was about 16.6%.

Screening of 35delG, W24X, W77X and Q124X Variants in connexin 26 gene associated with Non-Syndromic Hearing Impairment

Introduction: Hearing deficiency is the most common sensory incompetence that affects millions of people all over the world. Profound Deafness is a common disorder that occurs in 1:1000 neonates and the cause is hereditary. Gap Junction Beta2 (GJB2) gene which codes for connexion 26 protein is implicated in hearing. Mutation or defect in this gene leads to the most common form of the congenital disorder called Deaf Mutism (DM). The present study aims to screen the common variants associated with hearing impairment. Objective: To Screen common variants 35delG, W24X, W77X, and Q124X in connexin 26 gene of the North Karnataka population. Methods: 613 deaf-mute children from districts of North Karnataka were screened and 184 Non Syndromic Sensorineural Hearing-impaired children were included in the study. Genomic DNA was isolated using peripheral blood. Our gene of interest was amplified by Polymerase Chain Reaction (PCR) using target-specific primers. The PCR product was subjected to sequencing to analyze the mutations. Result: In our population prevalence of W24X, W77X variants were 25% & 5.4% respectively and 35 del G and Q124X mutations were absent. Along with this common variant, I33T (5.4%) and R127H (14.6%) were also observed. Conclusion: above findings suggests that Gap junction Beta 2 gene plays a crucial role in hearing impairment. Mutation analysis of the entire GJB2 gene in our population may help to define the etiology of deafness as well as the development of population-specific prenatal and perinatal genetic markers.

Using In Situ Padlock Probe Technology to Detect mRNA Splice Variants in Tumor Cells.

Advanced prostate cancer (PC) patients commonly receive anti-hormonal drugs targeting the androgen receptor (AR) signaling pathways. However, almost all patients acquire therapy resistance that can be caused by AR amplification or expression of AR splice variant 7 (AR-V7). Therefore, AR-V7 and AR expression are potential biomarkers for early detection of therapy resistance. Here, we present our padlock probe (PLP)-based approach for the in situ detection of AR full length, AR-V7, and prostate-specific transcripts in PC cell lines, which is applicable for circulating tumor cells (CTCs) isolated from cancer patients. First, PC cell lines are seeded on glass slides. Then, cDNA is created using target-specific reverse transcription primers. PLPs are hybridized to the cDNA and ligated to form circular single-stranded DNA molecules. The PLP sequence is ligated and amplified by rolling circle amplification and the resulting rolling circle products can be detected using fluorescently labeled probes. Quantification can be automated using the image analysis software CellProfiler.

The Detection of Minor Clones with Somatic KIT D816V Mutations Using Droplet Digital PCR in Pediatric De Novo AML: AML-05 Trial from the Japanese Pediatric Leukemia/Lymphoma Study Group

Introduction Recent medical advances and development of comprehensive genetic understanding dramatically improve the clinical outcome of whole pediatric cancers, particularly in pediatric acute lymphoblastic lymphoma. However, approximately 50% of patients have disease relapse, and overall survival (OS) of pediatric acute myeloid leukemia (AML) is less than 70% as of the major therapeutic challenges. AML is caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expressions, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. AML with RUNX1-RUNX1T1 gene fusions are generally classified as a low-risk group and resulted in favorable prognosis. However approximately 30% of the patients relapsed within 3 years. Conversely, KIT mutations were found in approximately 30% of AML cases with RUNX1-RUNX1T1 and thought to be a risk factor for relapse, particularly when occurring in D816V within KIT exon 17. Recently, droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, has been used to determine low-prevalence somatic mutations that were not detectable using Sanger sequencing. It shows the possibility that there are some of Pediatric AML cases which were not detected minor clones with somatic KIT mutation by using ordinary PCR. In this study, we explored KIT D816V mutations including the cases which are not detected by Sanger sequencing and found the prognosis of them by using Japanese pediatric AML cases. Methods We reanalyzed somatic KIT mutations (p.D816V) in the DNA extracted from 335 pediatric AML patients with RUNX1-RUNX1T1 who participated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial using ddPCR . In this trial, we conducted the tests as follows,: PCR mixture containing 10 μL 2x ddPCR Supermix for probes, 900 nM target-specific PCR primers, and 250 nM mutant-specific (FAM) and wild-type-specific (HEX) probes.20 µL of PCR mixture and 70 μL Droplet generation oil were mixed, and droplet generation was performed using a Bio-Rad QX100 Droplet Generator. The droplet emulsion was thermally cycled in the following conditions: denaturing at 95 °C for 10 min, 40 cycles of PCR at 94 °C for 30 s and at 57 °C for 2 min, and a final extension at 98 °C for 10 min. PCR amplification in the droplets was confirmed using Bio-Rad QX200 Droplet Reader. ThresholdThe threshold was determined by comparing the non-template ddPCR results. All the data were evaluated above the threshold. We also performed targeted gene mutation analysis of KIT in all patients using Sanger sequencing. Results We identified 24 KIT D816V mutations (7.2%) in the 335 pediatric AML. Variant allele frequency (VAF) was 0.1%-46.9%.It is noteworthy that 12 out of 24 KIT D816V mutations were undetected in our previous study using Sanger sequencing. Fourteen out of these 24 patients were AML with RUNX1-RUNX1T1, 5 cases with inv(16), and 5 cases with other alterations. Ten of the 14 RUNX1-RUNX1T1 (71%) patients were newly identified using ddPCR. Six of these 14 RUNX1-RUNX1T1 patients had relapsed, and D816V mutations were only detected using ddPCR in 4 of these 6 relapsed cases. The mean VAF of KIT D816V was 3.8% (0.1%-13.4%) in the 10 undetected patients with RUNX1-RUNX1T1. Two of the 5 patients with inv(16) were newly identified, and 1 had relapsed. All 5 cases with other alterations were already identified using Sanger sequencing. The mean VAF of KIT D816V in the 3 patients with inv(16) was 44.8% (42.7%-46.9%), detected using Sanger sequencing, whereas the mean VAF of KIT D816V was 8.6% (6.5% and 10.7%) in the undetected patients with inv(16). The mean VAF of KIT D816V with other alterations was 28.1% (16.2%-42.9%). Conclusion We identified 12 KIT D816V mutations using ddPCR that were undetected using Sanger sequencing. ddPCR may be useful for detecting accurate frequencies of mutations that were undetected using Sanger sequencing. Potential co-existing gene mutations may contribute different significance of leukemogenesis and relapse. Disclosures Ito: Asahi Kasei Pharma: Consultancy, Other: Grants; Astellas Pharma: Consultancy, Other: Grants; Teijin: Other: Grants; Novartis Pharma: Consultancy; Zenyaku Kogyo Co. Ltd: Consultancy; Chugai Pharma: Consultancy, Other: Grants; Japan Blood Products Organization: Other: Grants; Pfizer inc.: Other: Grants.

A portable detection assay for Apple stem pitting virus using reverse transcription-recombinase polymerase amplification

A molecular diagnostic assay for the rapid, sensitive and specific detection of Apple stem pitting virus (ASPV) in infected samples, utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 42 °C and the designed target-specific primers, was developed. The RT-RPA assay was able to be used in ASPV-infected leaves, rootstocks and fruits. Sensitivity tests, using ASPV transcripts, showed that the RT-RPA with the ASPV-specific primers was more sensitive than the conventional RT-PCR, with a detection limit of 1 fg/μL of RNA. In addition, the reaction time for the amplification of ASPV was shortened to as little as 1 min. The assay was highly specific and did not give a positive reaction to other viruses infecting pears. Moreover, the amplified genomic fragment of ASPV produced by the assay could be determined within 4 min using a portable capillary gel electrophoresis system. The entire process, excluding the extraction of total RNA, could be completed in 5 min using portable equipment in the field. This is the first report of utilizing an RT-RPA assay to detect a pear tree virus and the assay could be used both in the laboratory and in the field for ASPV detection.

Epichloe novae-zelandiae, a new endophyte from the endemic New Zealand grass Poa matthewsii

ABSTRACTEpichloe endophytes (Clavicipitaceae) infect pooid grass genera worldwide but predominantly in the Northern Hemisphere, but appear to be rare in native grasses of the Southern Hemisphere. Because of benefits that hosts may receive from the symbiosis, Epichloe endophytes have been extensively studied and are considered important components of sustainable agriculture. There are only a few studies available on the incidence of endophyte infection in grasses of the Southern Hemisphere and most grass species have never been examined. Here we report on a survey of native grasses of New Zealand including 25 endemic or indigenous species. We sampled up to 10 plants per species at different sites from both the North and South Island of New Zealand and examined tissues microscopically for endophyte infection. Overall, only two species were found to be infected, Poa matthewsii (Matthew's bluegrass) and Dichelachne micrantha (short-hair plume grass). Based on analyses of tefA and tubB genes, both endophytes were found to be interspecific hybrids. The endophyte of the new host D. micrantha was previously described as Epichloe australiensis, while the endophyte of P. matthewsii is a new species named here E. novae-zelandiae. The new species is a hybrid derived from E. amarillans, E. bromicola and E. typhina subsp. poae. Alkaloid analyses in planta suggested that E. novae-zelandiae can produce small amounts of peramine, early pathway indole-diterpenes and ergot alkaloids, but no lolines or lolitrems. Target specific primers suggested the presence of genes for ergot alkaloids and peramine, but genes of only early pathway steps for the other alkaloids. Furthermore, genes for both mating type idiomorphs (MTA and MTB) were present, a single copy of MTA and two copies of MTB. Endophytes of native grasses may provide a genetic resource that could be exploited for developing pasture grass cultivars with improved performance.

Abstract 3532: Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow

Abstract Tumor mutational burden (TMB) is currently of high interest in the field of immuno-oncology due to its correlation with patient response to checkpoint inhibitor chemotherapy. Traditionally, TMB is calculated using whole exome sequencing; however, targeted sequencing approaches provide better coverage of the genetic regions of interest at lower costs. While hybrid-capture based target enrichment methods are well-established, the 2-5 day workflows are time consuming and require specialized equipment and highly trained operators. Here we present an ultrafast, 4-hour method for preparing target enriched NGS libraries for assessing TMB that would streamline and lower the cost of immuno-oncology studies. This multiplex-PCR-based technology provides a highly efficient, accurate and robust method for unbiased enrichment of tens of thousands of target regions while minimizing non-specific primer-primer interactions and GC bias and maximizing coverage uniformity. We demonstrate this using a highly-multiplexed prototype NGS panel that contains ~20,000 amplicons and covers 355 genes for assessment of TMB. Sequencing-ready NGS libraries were prepared using the Paragon Genomics CleanPlex® target enrichment technology. The 3-step workflow combines target enrichment and NGS library preparation. The protocol includes an ultra-high multiplex PCR step to amplify regions of interest with target-specific primers, a background cleaning step to remove non-specific PCR products, and a final PCR to add Illumina sequencing adapter and sample indexes. Libraries were made using 20ng of genomic DNA and sequenced on an Illumina NextSeq® platform. Sequencing metrics such as on-target rates were calculated, and variants were identified using Paragon Genomics’ variant calling algorithm. Using the CleanPlex technology, this prototype TMB panel exhibits >95% uniformity at 0.2X mean, limited GC bias, and >94% detection rate for mutants with 5% allele frequencies. The CleanPlex background cleaning step was essential for removing the undesirable byproducts of the multiplexed reaction step. CleanPlex technology demonstrates that it is capable of creating high quality amplicon libraries with high uniformity, low GC bias, and sensitive variant calling even in ultra-multiplexed libraries with ~20,000 amplicons with a workflow under 4-hours. Citation Format: Kathryn E. Pendleton, Yang L. Liu, Lifeng Lin, Lucie S. Lee, Jeffery Liu, Guoying Liu, Zhitong Liu. Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3532.

Evaluating methods for Avian avulavirus-1 whole genome sequencing.

BackgroundAvian avulavirus-1 (AAvV-1, previously Newcastle Disease Virus) is responsible for poultry and wild birds' disease outbreaks. Numerous whole genome sequencing methods were reported for this virus. These methods included cloning, specific primers amplification, shotgun PCR approaches, Sequence Independent Single Primer Amplification and next generation sequencing platform kits.MethodsThree methods were used to sequence 173 Israeli Avian avulavirus-1 field isolates and one vaccine strain (VH). The sequencing was performed on Proton and Ion Torrent Personal Genome Machine and to a lesser extent, Illumina MiSeq and NextSeq sequencers. Target specific primers (SP) and Sequence Independent Single Primer Amplification (SISPA) products sequenced via the Ion torrent sequencer had a high error rate and truncated genomes. All the next generation sequencing platform sequencing kits generated high sequence accuracy and near-complete genomic size.ResultsA high level of mutations was observed in the intergenic regions between the avian avulavirus-1 genes. Within genes, multiple regions are more mutated than the Fusion region currently used for typing.ConclusionsOur findings suggest that the whole genome sequencing by the Ion torrent sequencing kit is sufficient. However, when higher fidelity is desired, the Illumina NextSeq and Proton torrent sequencing kits were found to be preferable.

Identification of canine norovirus in dogs in South Korea

BackgroundCanine noroviruses (CaNoVs) are classified into genogroups GIV, GVI, and GVII and have been detected in fecal samples from dogs since their first appearance in a dog with enteritis in Italy in 2007. CaNoVs may be a public health concern because pet animals are an integral part of the family and could be a potential reservoir of zoonotic agents. Nonetheless, there was no previous information concerning the epidemiology of CaNoV in South Korea. In the present study, we aimed to detect CaNoV antigens and to investigate serological response against CaNoV in dogs.ResultsIn total, 459 fecal samples and 427 sera were collected from small animal clinics and animal shelters housing free-roaming dogs in geographically distinct areas in South Korea. For the detection of CaNoV, RT-PCR was performed using target specific primers, and nucleotide sequences of CaNoV isolates were phylogenetically analyzed. Seroprevalence was performed by ELISA based on P domain protein. CaNoVs were detected in dog fecal samples (14/459, 3.1%) and were phylogenetically classified into the same cluster as previously reported genogroup GIV CaNoVs. Seroprevalence was performed, and 68 (15.9%) of 427 total dog serum samples tested positive for CaNoV IgG antibodies.ConclusionThis is the first study identifying CaNoV in the South Korean dog population.

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.

A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

BackgroundMost current methods for constructing guide RNAs (gRNA) for the CRISPR/Cas9 genome editing system, depend on traditional cloning using specific type IIS restriction enzymes and DNA ligation. These methods consist of multiple steps of cloning, and are time consuming, resource intensive and not flexible. These issues are particularly exacerbated when multiple guide RNAs need to be assembled in one plasmid such as for multiplexing or for the paired nickases approach. Furthermore, identification of functional gRNA clones usually requires expensive in vitro screening. Addressing these issues will greatly facilitate usage and accessibility of CRISPR/Cas9 genome editing system to resource-limited laboratories.ResultsTo improve efficiency of cloning multiple guide RNAs for the CRISPR/Cas9 system, we developed a restriction enzyme- and ligation-independent strategy for cloning gRNAs directly in plant expression vectors in one step. Our method relies on a negative selection marker and seamless cloning for combining multiple gRNAs directly in a plant expression vector in one reaction. In addition, using the Agrobacterium-mediated transient assays, this method provides a simple in planta procedure for assaying the effectiveness of multiple gRNAs very rapidly.ConclusionsFor a fraction of resources used in the type IIS restriction enzyme-based cloning method and in vitro screening assays, the system reported here allows efficient construction and testing several ready-to-transfect gRNA constructs in < 3 days. In addition, this system is highly versatile and flexible, and by designing only two additional target-specific primers, multiple gRNAs can be easily assembled in any plasmid in a single reaction.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen.

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population.

BioSpotlight / Citations

BioTechniquesVol. 63, No. 2 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo, & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author, & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114572AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Taking charge of extracellular vesiclesExtracellular vesicles (EVs) play important roles in long-distance intercellular communication and are potentially useful as biomarkers. EVs have been isolated from body fluids by centrifugation, size exclusion chromatography, filtration, precipitation, and affinity purification; however, these methods vary in the extent of contamination of the isolated EVs with non-EV protein complexes and RNAs. In this issue of BioTechniques, Maja Kosanović and her colleagues at the University of Belgrade present a new method for isolating EVs that reduces such contamination. Given that they are negatively charged due to their surface molecules, the team tested ion-exchange chromatography (IEC) as an approach for purifying EVs. Using amniotic fluid as the source of EVs, they compared the gold-standard method, consisting of ultracentrifugation followed by sucrose density gradient centrifugation, with their novel method of ultracentrifugation followed by IEC. As demonstrated by protein electrophoresis and immuno- and lectin blotting, the use of IEC significantly reduced the contamination seen with the gold-standard method.See “Ion-exchange chromatography purification of extra-cellular vesicles” on page 65.Rapid and no-wash live-cell stainingLive-cell fluorescent dyes are critical tools for cell biology research, allowing the visualization and tracking of cells in various assays. In this issue, Alexey A. Pakhomov and his colleagues at the Russian Academy of Sciences describe a new fluorescent dye for rapid, no-wash staining of live cells. This dye is derived from boron dipyrromethene (BODIPY), a molecule with numerous other derivatives that are used as fluorescent sensors and for conjugation to biomolecules. Some of these derivatives are lipophilic, binding to the plasma and subcellular membranes, and they tend to aggregate in polar media such as the cytosol and extracellular space, which results in aggregation-caused quenching. The team tested these derivatives for use in live-cell staining and demonstrated that one of them immediately stained the plasma and subcellular membranes of live mammalian cells with high contrast, without the need for washing. In fact, washing the stained cells with PBS removed the dye, allowing the cells to be stained with another dye of the same color.See “BODIPY-based dye for no-wash live-cell staining and imaging” on page 77.Two-tailed miRNA quantificationThe analysis of microRNAs (miRNAs) is challenging given the short sequence lengths of these molecules. In an attempt to design a more specific and sensitive assay to quantify miRNA expression patterns, Androvic et al. developed a new method they named Two-tailed RT-qPCR. This technique uses a unique primer design incorporating two specific hemiprobes, which are complementary to the miRNA of interest and separated by a tether that can fold into a hairpin to prevent non-specific inter-actions. The reverse transcription reaction to generate cDNA is primed from the 3´-end hemiprobe, while the 5´-end hemiprobe is displaced. After reverse transcription, the resulting cDNA is quantified using two different target-specific primers. The authors showed that Two-tailed RT-qPCR has the sensitivity to detect as few as 10 target miRNA molecules while providing a dynamic range of 7 logs. This new approach should be of particular interest to those seeking highly accurate miRNA quantification.Androvic, P. et al. 2017. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Res. doi:10.1093/nar/gkx588Massive cell separationSingle-cell sequencing has become commonplace in life science research. By studying single cells, scientists are learning about cellular heterogeneity, lineage determination, and hierarchical relationships. In a new article in BMC Genomics, Goldstein et al. take single-cell sequencing high-throughput with their description of a massively parallel single-cell profiling pipeline. The authors took advantage of the ICELL8 system, which enables the separation of cell mixtures into 5184 nanowells and then uses imaging to locate wells possessing only a single cell. Once a nanowell containing a single cell is identified, in-well mRNA isolation and barcoded-cDNA generation is performed, after which the resulting cDNA from multiple wells is pooled for sequence analysis. Using the pipeline, the authors were able to sequence transcriptomes from more than 1000 cultured human and mouse cells, along with nearly 500 mouse pancreatic islet cells.Goldstein, L.D. et al. 2017. Massively parallel nanowell-based single-cell gene expression profiling. BMC Genomics. 18:519.FiguresReferencesRelatedDetails Vol. 63, No. 2 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print August 2017 Information© 2017 Author(s)PDF download