Rounds Of Amplification Research Articles

A-248 Assessment of Nanopore Sequencing for HIV Genotyping: A Comparative Study with Sanger Sequencing

Abstract Background The HIV drug resistance test, conducted through genotyping of the Human Immunodeficiency Virus (HIV), is a critical tool for assessing drug resistance in virus-infected patients and is commonly performed using Sanger Sequencing methodology (first-generation sequencing). Nanopore sequencing (third-generation sequencing) is a new technology that started to be commercialized a few years ago and has recently undergone significant updates with the new Q20+ chemistry. Therefore, this study aims to evaluate the potential use of nanopore sequencing (MinION) for HIV genotyping. Methods The genomic region encoding HIV protease and transcriptase was amplified by nested PCR using primer pairs modified with unique barcodes in the second round of amplification. Three libraries were assembled, including samples from 22 HIV-positive patients, one negative control (negative control patient), and one no-template control (NTC) per library, totaling 66 HIV-positive samples. The libraries were constructed using the SQK-LSK114 kit and sequenced on a MinION platform using the new Q20+ chemistry in flowcells or flongle R10.4.1. The sequences were assembled and analyzed using HIVdb-NGS (Stanford). Validation tests, such as accuracy and precision, were conducted following WHO recommendations for HIV genotyping. The same samples were also sequenced using the Sanger method for comparison. Results 62 HIV sequences were generated by the MinION and analyzed in the described pipeline. The average sequence coverage per sample was 25,866 x. 41 sequences were classified as type B (63.08%), 12 as type C (18.46%), 8 as type F (12.31%), three as CRF02_AG (4.62%), and 1 as a recombinant B+C (1.54%). All identified subtypes were consistent with those found by the Sanger method. The average similarity between the sequences of each sample (MinION vs Sanger) was 98.68% (minimum 94.96%, maximum 99.95%). Basecalling similarity at relevant positions (associated with resistance) was above 98% in 59 out of 62 samples (95.16%), meeting the acceptance criteria recommended by the WHO. Conclusions Our results suggest that nanopore sequencing (utilizing Q20+ chemistry) can replace Sanger sequencing for HIV genotyping, which is used to monitor the effectiveness of anti-HIV drugs. This transition will allow for a reduction in test costs in laboratories with high demand for HIV drug resistance testing. Additionally, the use of modified primers proposed here eliminates the need for commercial barcodes, resulting in reduced processing time and improved throughput.

Leishmania in skin of Rattus rattus from the urban area in the Corrientes city, Argentina

Objective: the objective of the present work was the detection of Leishmania sp. in Rattus rattus tail base skin through polymerase chain reaction (PCR) techniques. Material and Method: We analyzed 45 Rattus rattus skin samples from the urban area of the city of Corrientes in Argentina. The Leishmania spp detection was performed by nested PCR technique. Results: Leishmania DNA was detected in 22 samples out of 45 processed (49%) in the first round of amplification, and in 14 samples (31%) in the second round (31%). Conclusions: These results contribute to increase the existing information in our region on the possible relationship between Leishmania and Rattus rattus, considering the high prevalence found in skin added to the total absence of lesions. Other aspects should be further studied to establish the role of these animals in the epidemiological chain of the disease in an urban area endemic to leishmaniasis in other animal species.

Synthesis of dual models multivalent activatable aptamers based on HCR and RCA for ultrasensitive detection of Salmonella typhimurium

Aptamers have superior structural properties and have been widely used in bacterial detection methods. However, the problem of low affinity still exists in complex sample detection. In contrast, hybridization chain reaction (HCR)-based model I and rolling circle amplification (RCA)-based model II multivalent activatable aptamers (multi-Apts) can fulfill the need for low-cost, rapid, highly sensitive and high affinity detection of S. typhimurium. In our research, two models of multi-Apts were designed. First, a monovalent activatable aptamer (mono-Apt) was constructed by fluorescence resonance energy transfer (FRET) with an S. typhimurium aptamer and its complementary chain of BHQ1. Next, the DNA scaffold was obtained by HCR and RCA, and the multi-Apts were obtained by self-assembly of the mono-Apt with a DNA scaffold. In model I, when target was presented, the complementary chain BHQ1 was released due to the binding of multi-Apts to the target and was subsequently adsorbed by UIO66. Finally, a FRET-based fluorescence detection signal was obtained. In mode II, the multi-Apts bound to the target, and the complementary chain BHQ1 was released to become the trigger chain for the next round of amplification of HCR with a fluorescence detection signal. HCR and RCA based multi-Apts were able to detect S. typhimurium as low as 2 CFU mL−1 and 1 CFU mL−1 respectively. Multi-Apts amplification strategy provides a new method for early diagnosis of pathogenic microorganisms in foods.

Alternative amplicon-PCR protocol for maximizing bacterial and fungal sequencing in low-biomass samples

Determining bacterial and fungal communities from low-biomass samples remains a challenge for high-throughput sequencing. Due to the low microbial load and host contamination, some sites, including the female upper reproductive tract and the lower respiratory tract, were even considered sterile until recent years. Despite efforts to improve sampling and DNA isolation protocols, some samples provide insufficient microbial DNA input for library preparation and sequencing. Herein, we propose an alternative amplicon-PCR protocol to be used in bacterial and fungal sequencing in low-biomass samples, targeting 16S-rDNA and the internal transcribed spacer region (ITS), respectively. Similar to a nested-PCR, we performed two sequential PCR reactions to maximise the target amplicon. We compared metagenomic results from the original Illumina protocol (Protocol 1 – P1) and the alternative one (Protocol 2 – P2), using a mock community and clinical samples with different microbial loads. Our findings showed no significant differences in data generated by P1 and P2, indicating that the second amplification round does not bias the microbiota diversity rates. Thus, the alternative protocol can be applied for low-biomass samples when the original protocol results in spurious output, preventing library preparation and sequencing.

Evaluation of the Thermo Fisher Oncomine Myeloid MRD DNA Assay on the Ion Torrent Platform

Introduction Malignant myeloid diseases are a group of blood cancers that arise from genetic changes in hematopoietic cells in the bone marrow or peripheral blood. The presence of measurable residual disease (MRD), or detection of abnormal cells in the body following completion of therapy, is associated with a higher risk of disease relapse, and decreased overall survival (Schuurhuis 2018). Next-generation sequencing (NGS) can be leveraged as a high-sensitivity, minimally invasive method for detection of MRD. The Ion Torrent Oncomine Myeloid MRD Assay (Myeloid MRD) is a targeted NGS assay designed for the detection of single nucleotide variants (SNVs) and insertions/deletions in a curated selection of clinically relevant myeloid disease-related genes at low limits of detection. Assay performance was evaluated using normal and diseased clinical blood and bone marrow samples and NGS reference materials. Methods The Myeloid MRD DNA panel targets hotspot regions across 33 genes and uses Ion AmpliSeq HD technology with unique molecular tagging to suppress sequencing artifacts and improve analytical sensitivity. Briefly, DNA was extracted from whole blood or bone marrow samples and 120 ng of nucleic acid was used as input for the assay. The DNA was divided between 2 polymerase chain reactions (PCR), where it served as the template for target amplifications, before combining reaction products by sample. Library preparation followed the standard Ion AmpliSeq HD protocol, where amplicons were partially enzymatically digested, and a second round of amplification was performed using barcoded primers. Following bead purification, the final library was measured by quantitative PCR (qPCR) to inform final library loading onto the sequencer. Six libraries were pooled and sequenced on an Ion 550 chip using the Ion GeneStudio S5 System. Primary and secondary analyses were performed using the Torrent Suite software, and results were exported to the Ion Reporter software for variant calling. From the variant calling results, a probit model was utilized to determine the variant allele frequency (VAF) at which the assay achieved 90% power to call an expected variant, defining the limit of detection (LoD). Other parameters evaluated included analytical sensitivity, analytical specificity, positive predictive value (PPV), accuracy, and precision. Results Overall, consistent, high quality on-target reads were observed across 22 sequencing runs with a median Ion Sphere Particle (ISP) loading of 90%, usable sequence of 59%, and over 130 million total reads per chip. When evaluating individual samples according to the vendor-recommended number of mapped reads (> 10 million), 98.3% of samples tested passed quality control (QC). Additional sample QC metrics established during assay validation included thresholds of > 80% reads on-target, > 80% uniformity of base coverage, and a mean read length of at least 80 base pairs. The estimated LoD for the assay was 0.1% VAF for small variants (SNVs, insertions/deletions).Above the LoD, the assay demonstrated 96.03% sensitivity, 99.89% accuracy of base-calling, 100% specificity, and 98.83% PPV.Additionally,a comparison was performed usingmatched whole blood and bone marrow samples collected from patients with acute myeloid leukemia (AML) to evaluate the concordance of variant calling. It was determined that the same variants were identified across both sample types, although at higher allele frequency in bone marrow compared to whole blood. Conclusion This evaluation of the Myeloid MRD assay demonstrates that it is a precise and accurate assay with sensitivity that is acceptable for MRD analysis. Notably, variant detection was concordant between whole blood and bone marrow sample types collected from AML patients, with bone marrow providing improved sensitivity for low-frequency variant detection. The user-friendly workflow and integrated informatics pipeline offer opportunities for streamlined testing and interpretation of results. The Ion Torrent Myeloid MRD DNA assay is a powerful new tool for MRD detection across a growing myeloid disease target landscape.

Selection of mutant Listeria phages under food-relevant conditions can enhance application potential.

Bacteriophages are viruses that infect and kill bacteria. Currently, phage products are available for the control of the pathogen Listeria monocytogenes in food products in the United States. In this study, we explore whether experimental evolution can be used to generate phages with improved abilities to function under specific food-relevant conditions. Ultra-pasteurized oat and whole milk were chosen as test matrices as they represent different food groups, yet have similar physical traits and macronutrient composition. We showed that (i) wild-type phage LP-125 infection kinetics are different in the two matrices and (ii) LP-125 has a significantly higher burst size in oat milk. From this, we attempted to evolve LP-125 to have improved infection kinetics in whole milk. Ancestral LP-125 was passaged through 10 rounds of amplification in milk conditions. Plaque-purified DNA samples from milk-selected phages were isolated and sequenced, and mutations present in the isolated phages were identified. We found two nonsynonymous substitutions in LP125_108 and LP125_112 genes, which encode putative baseplate-associated glycerophosphoryl diester phosphodiesterase and baseplate protein, respectively. Protein structural modeling showed that the substituted amino acids in the mutant phages are predicted to localize to surface-exposed helices on the corresponding structures, which might affect the surface charge of proteins and their interaction with the bacterial cell. The phage containing the LP125_112 mutation adsorbed significantly faster than the ancestral phage in both oat and whole milk. Follow-up experiments suggest that fat content may be a key factor for the expression of the phenotype of this mutation. IMPORTANCE Bacteriophages are one of the tools available to control the foodborne pathogen, Listeria monocytogenes. Phage products must work under a broad range of food conditions to be an effective control for L. monocytogenes. Here, we show that the experimental evolution of phages can be used to generate new phages with phenotypes useful under specific conditions. We used this approach to select for a mutant phage that more efficiently binds to L. monocytogenes that is grown in whole milk and oat milk. We show that the fat content of these milks is necessary for the expression of this phenotype. Our findings show that experimental evolution can be used to select for improved phages with better performance under specific conditions. This approach has the potential to support the development of condition-specific phage-based biocontrols in the food industry.

A targeted sequencing extension for transcript genotyping in single-cell transcriptomics.

As no existing methods within the single-cell RNA sequencing repertoire combine genotyping of specific genomic loci with high throughput, we evaluated a straightforward, targeted sequencing approach as an extension to high-throughput droplet-based single-cell RNA sequencing. Overlaying standard gene expression data with transcript level genotype information provides a strategy to study the impact of genetic variants. Here, we describe this targeted sequencing extension, explain how to process the data and evaluate how technical parameters such as amount of input cDNA, number of amplification rounds, and sequencing depth influence the number of transcripts detected. Finally, we demonstrate how targeted sequencing can be used in two contexts: (1) simultaneous investigation of the presence of a somatic variant and its potential impact on the transcriptome of affected cells and (2) evaluation of allele-specific expression of a germline variant in ad hoc cell subsets. Through these and other comparable applications, our targeted sequencing extension has the potential to improve our understanding of functional effects caused by genetic variation.

Automated System for Multiplexing Detection of COVID-19 and Other Respiratory Pathogens.

Infectious diseases are global health challenge, impacted the communities worldwide particularly in the midst of COVID-19 pandemic. The need of rapid and accurate automated systems for detecting pathogens of concern has always been critical. Ideally, such systems shall detect a large panel of pathogens simultaneously regardless of well-equipped facilities and highly trained operators, thus realizing on-site diagnosis for frontline healthcare providers and in critical locations such as borders and airports. Avalon Automated Multiplex System, AAMST, is developed to automate a series of biochemistry protocols to detect nucleic acid sequences from multiple pathogens in one test. Automated processes include isolation of nucleic acids from unprocessed samples, reverse transcription and two rounds of amplifications. All procedures are carried out in a microfluidic cartridge performed by a desktop analyzer. The system was validated with reference controls and showed good agreement with their laboratory counterparts. In total 63 clinical samples, 13 positives including those from COVID-19 patients and 50 negative cases were detected, consistent with clinical diagnosis using conventional laboratory methods. The proposed system has demonstrated promising utility. It would benefit the screening and diagnosis of COVID-19 and other infectious diseases in a simple, rapid and accurate fashion. Clinical and Translational Impact Statement- A rapid and multiplex diagnostic system proposed in this work can clinically help to control spread of COVID-19 and other infectious agents as it can provide timely diagnosis, isolation and treatment to patients. Using the system at remoted clinical sites can facilitate early clinical management and surveillance.

Fast Black-Box Quantum State Preparation

Quantum state preparation is an important ingredient for other higher-level quantum algorithms, such as Hamiltonian simulation, or for loading distributions into a quantum device to be used e.g. in the context of optimization tasks such as machine learning. Starting with a generic "black box" method devised by Grover in 2000, which employs amplitude amplification to load coefficients calculated by an oracle, there has been a long series of results and improvements with various additional conditions on the amplitudes to be loaded, culminating in Sanders et al.'s work which avoids almost all arithmetic during the preparation stage.In this work, we construct an optimized black box state loading scheme with which various important sets of coefficients can be loaded significantly faster than in O(N) rounds of amplitude amplification, up to only O(1) many. We achieve this with two variants of our algorithm. The first employs a modification of the oracle from Sanders et al., which requires fewer ancillas (log2⁡g vs g+2 in the bit precision g), and fewer non-Clifford operations per amplitude amplification round within the context of our algorithm. The second utilizes the same oracle, but at slightly increased cost in terms of ancillas (g+log2⁡g) and non-Clifford operations per amplification round. As the number of amplitude amplification rounds enters as multiplicative factor, our black box state loading scheme yields an up to exponential speedup as compared to prior methods. This speedup translates beyond the black box case.

Can cloning and sequencing help to genotype positive Toxoplasma gondii clinical samples? Results and validation using SAG3 as a model.

Genotyping of T. gondii in human cases is relevant to understand the transmission patterns and epidemiology of this parasitosis. However, this genetic characterization can be hampered by the difficulty of isolating the parasite from mild or asymptomatic cases and by the detection efficiency of molecular assays such as the multilocus nested-polymerase chain reaction-restriction fragment length polymorphism (Mn-PCR-RLFP). To propose an alternative for the genotyping of positive clinical samples of T. gondii with a low amount of the parasite DNA mixed within the host DNA or mixed infections, we carried out this study to validate the sequences of the SAG3 gene of T. gondii obtained after two rounds of amplification cloned into a bacterial model, thereby achieving the separation and identification of more than one genotype of T. gondii. Also, the detection limit of the parasite DNA and the fidelity of the reagents used in the nested PCR-RFLP in artificial clinical samples by sequencing were determined. T. gondii DNA was detected from 6.25ng of DNA and 200 parasites/mL of blood. The fidelity of the AmpliTaq Gold™ polymerase after 65cycles of amplification was 100%. Denaturation of the products obtained after two rounds of nested PCR amplification showed no evidence of chimera or artifact production. The cloning efficiency was 97.5% (39/40 clones), and none of the experiments produced recombinant sequences. Thus, the generation of chimeras with this methodology could be ruled out. Genotyping of clinical samples is important because there is no strain selection bias, as can occur in the bioassay (where more virulent strains can be selected over nonvirulent strains), and therefore, mixed infections can be detected through cloning and sequencing. Furthermore, these two techniques could be useful tools to genotype weak amplicons of any T. gondii gene obtained during nested PCR.

Comparison of three PCR‐based methods to detect Loa loa and Mansonella perstans in long‐term frozen storage dried blood spots

Loa loa and Mansonella perstans are two very common filarial species in Africa. Although microscopy is the traditional diagnostic method for human filariasis, several polymerase chain reaction (PCR) methods have emerged as an alternative approach for identifying filarial parasites. The aim of this study is to compare three molecular methods and decide which is the most suitable for diagnosing human loiasis and mansonellosis in non-endemic regions using dried blood spot (DBS) as a medium for sample collection and storage. A total of 100 DBS samples, with their corresponding thin and thick blood smears, were selected for this study. Microscopy was used as the reference method to diagnose and calculate the microfilaraemia. Filarial DNA was extracted using the saponin/Chelex method and the DNA isolated was assayed by Filaria-real time-PCR, filaria-nested PCR, and cytochrome oxidase I PCR. All PCR products were subsequently purified and sequenced. The statistical values for each molecular test were calculated and compared. Overall, 64 samples were identified as negative by all tests and a further 36 samples were positive by at least one of the methods used. The sensitivity and specificity were similar for the different molecular methods, all of which demonstrated good agreement with microscopy. Based on this study, and from a practical point of view (single and short amplification round), the optimal technique for diagnosing filarial infection in non-endemic regions is filaria-real time-PCR, which presents high sensitivity and specificity and is also able to detect a wide range of human filariae.

In Vitro Selection of RNAs That Can Detect Hypoxanthine: A Surprising Technical Issue

The goal of this project is to find RNA molecules that detect hypoxanthine with great sensitivity and specificity. Hypoxanthine is the product of a family of RNA editing enzymes called ADARs. A sensor that can detect hypoxanthine will facilitate the study of the regulation of ADAR activity. There are naturally occurring guanine sensors called guanine riboswitches. We are using in vitroselection to find variants of a guanine riboswitch that can detect hypoxanthine rather than guanine. While performing the selection experiment, we encountered a technical problem. Instead of isolating hypoxanthine sensors, a group of related aberrant RNAs dominated the selected RNA pool. These RNAs had many sequence changes and deletions compared to the sequence of the guanine riboswitch, and they could not detect hypoxanthine. The aberrant RNAs were produced not by the selection process, but as a consequence of multiple rounds of amplification. Here we report our attempts to understand why this happened and to prevent the formation of the aberrant RNAs.

New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly s...

Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence

Circle-to-circle amplification (C2CA) is a specific and precise cascade nucleic acid amplification method consisting of more than one round of padlock probe ligation and rolling circle amplification (RCA). Although C2CA provides a high amplification efficiency with a negligible increase of false-positive risk, it contains several step-by-step operation processes. We herein demonstrate a homogeneous and isothermal nucleic acid quantification strategy based on C2CA and optomagnetic analysis of magnetic nanoparticle (MNP) assembly. The proposed homogeneous circle-to-circle amplification eliminates the need for additional monomerization and ligation steps after the first round of RCA, and combines two amplification rounds in a one-pot reaction. The second round of RCA produces amplicon coils that anneal to detection probes grafted onto MNPs, resulting in MNP assembly that can be detected in real-time using an optomagnetic sensor. The proposed methodology was applied for the detection of a synthetic complementary DNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as 2019-nCoV) RdRp (RNA-dependent RNA polymerase) coding sequence, achieving a detection limit of 0.4 fM with a dynamic detection range of 3 orders of magnitude and a total assay time of ca. 100 min. A mathematical model was set up and validated to predict the assay performance. Moreover, the proposed method was specific to distinguish SARS-CoV and SARS-CoV-2 sequences with high similarity.

Study on the Effective Method of Optimizing PCR-DGGE by adding PCR enhancers

PCR-based denaturing gradient gel electronic (PCR-DGGE) has become a frequently used method in the determination of bacteria community in water, soil and other habitats. However, the precision and the integrality of microbiology community by this approach should be reinforced and the key for this is optimization on PCR for which is the basis of PCR-DGGE. Aiming at this, PCR enhancer combinations were applied in both two amplification rounds of PCR-DGGE in this study. The results showed that many new bands were produced and originally weak bands were intensified after the addition of PCR enhancer combinations, particularly DBD-2 (1% DMSO, 0.4 M Betaine, 1mM DTT) in PCR amplification buffer system. Virtually all newly appeared bands in DGGE were probably derived from the bacteria with high GC% content (60%). Thus the major optimization on PCR-DGGE was PCR enhancer may contribute to decreasing annealing temperature and improving the PCR product yields of the high-GC%-contented bacteria. In conclusion, PCR enhancers could remarkably improve the amplification efficacy, particularly for using the complex and tiny environmental DNA or weaker bands from DGGE as PCR template, and furthermore increase the detected bacterial species in PCR-DGGE. Therefore we strongly proposed that the PCR enhancer combination be routinely applied in PCR-DGGE for the bacterial community determination.

Click chemistry-based amplification and detection of endogenous RNA and DNA molecules in situ using clampFISH probes.

Direct labeling and measurement of gene expression in single cells show the tremendous variability otherwise hidden in bulk measurements. Single-molecule RNA fluorescence in situ hybridization (FISH) has become a mainstay in laboratories worldwide for measuring gene expression with precision. However, this method remains relatively low throughput because the total fluorescent signal produced is weak and requires long exposure times and high magnification microscopy, which limits the total number of cells sampled in each image. As such, it is experimentally difficult and time-consuming to sample a large enough population of cells to visualize and quantify specific gene expression of rare cells directly. Several FISH-based tools were recently developed that retain single-molecule sensitivity and specificity while greatly amplifying the fluorescent signal, thus making FISH-based analysis possible using standard microscopes with low magnification objectives. These tools have also enabled the detection of smaller and more specific targets like splice junctions or single nucleotide polymorphisms. Here we will describe one such tool, clampFISH, an oligonucleotide-based fluorescence amplification strategy for visualizing genomic loci and individual RNA transcripts in fixed cells. ClampFISH maintains specificity while amplifying fluorescent signals, making it amenable to high throughput assays such as low magnification microscopy, spatial transcriptomics, and flow sorting. The clampFISH technique involves probing the target RNA or DNA using a series of C-shaped oligonucleotide probes, each with a 3' azide and a 5' alkyne. Hybridization of the probe with the target nucleic acid brings the azide and the alkyne in close proximity, allowing for ligation via bioorthogonal click chemistry (CuAAC). As a result, the probe forms a closed loop around the target sequence, thus enabling stringent washes to remove nonspecific binding in further rounds of amplification and retention of signal throughout liquid handling steps. Iterative rounds of hybridization with C-shaped, fluorescently labeled probes exponentially amplify the fluorescent signal. ClampFISH is simple to implement and expands the utility of in situ hybridization for multiple high throughput techniques such as low magnification microscopy, flow cytometry, and sorting based on RNA expression levels.

A scalable suspension insect cell transfection method for production of baculoviruses with low amplification passages

Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 × 106 cells, the recombinant baculoviruses titer obtained with modified method was about 2 × 107 pfu/ mL in a total volume of 12 mL, which is scalable to 24 liters of 1 × 108 pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages.

Draft genome sequence of Solanum aethiopicum provides insights into disease resistance, drought tolerance, and the evolution of the genome.

BackgroundThe African eggplant (Solanum aethiopicum) is a nutritious traditional vegetable used in many African countries, including Uganda and Nigeria. It is thought to have been domesticated in Africa from its wild relative, Solanum anguivi. S. aethiopicum has been routinely used as a source of disease resistance genes for several Solanaceae crops, including Solanum melongena. A lack of genomic resources has meant that breeding of S. aethiopicum has lagged behind other vegetable crops.ResultsWe assembled a 1.02-Gb draft genome of S. aethiopicum, which contained predominantly repetitive sequences (78.9%). We annotated 37,681 gene models, including 34,906 protein-coding genes. Expansion of disease resistance genes was observed via 2 rounds of amplification of long terminal repeat retrotransposons, which may have occurred ∼1.25 and 3.5 million years ago, respectively. By resequencing 65 S. aethiopicum and S. anguivi genotypes, 18,614,838 single-nucleotide polymorphisms were identified, of which 34,171 were located within disease resistance genes. Analysis of domestication and demographic history revealed active selection for genes involved in drought tolerance in both “Gilo” and “Shum” groups. A pan-genome of S. aethiopicum was assembled, containing 51,351 protein-coding genes; 7,069 of these genes were missing from the reference genome.ConclusionsThe genome sequence of S. aethiopicum enhances our understanding of its biotic and abiotic resistance. The single-nucleotide polymorphisms identified are immediately available for use by breeders. The information provided here will accelerate selection and breeding of the African eggplant, as well as other crops within the Solanaceae family.

CLAmp‐seq: A Novel Amplicon‐Based NGS Assay with Concatemer Error Correction for Improved Detection of Actionable Mutations in Plasma cfDNA from Patients with NSCLC

AbstractHigh‐throughput sequencing of circulating cell‐free DNA (cfDNA) is critical for targeted therapy selection and monitoring in non‐small cell lung cancer (NSCLC) patients. However, low quantities of cfDNA in the blood and assay artifacts limit the sensitivity and specificity of next‐generation sequencing (NGS) test. This study develops a novel amplicon‐based NGS technology, CLAmp‐seq (circular ligation amplification and sequencing), with a concatemer‐based error correction strategy, which effectively removes polymerase errors from the first round of amplification. Analytical validation shows median detection rate of 100% at a mutant allele frequency (MAF) of 0.1% for 20 ng of input. The assay achieves 99.8% and 99.5% concordance of repeatability and reproducibility for 0.1% MAF, respectively, and both 100% for 0.5% MAF cfDNA standards. A comparative analysis of 134 NSCLC patient plasma samples demonstrates strong concordance (97.4%) and linearity (R2 = 0.95) between the CLAmp‐seq NGS readouts and droplet digital PCR (ddPCR) results. The detection of mutants in cfDNA obtained from pretreatment plasma samples shows 94.8% concordance with tissue genotyping results. No mutation is detected in noncancerous control samples. It shows that CLAmp‐seq NGS assay can offer an easy‐to‐use, fast, and accurate molecular diagnostic tool with multiplex capacity that is well‐suited for clinical in vitro diagnosis.

Amplification of RNA for Microarrays.

Gene expression profiling typically requires microgram quantities of mRNA, which can be difficult to obtain. In such cases, RNA must be amplified to have enough material for microarray labeling and hybridization. This protocol generates amplified antisense RNA (aRNA) from limited quantities of total RNA. It is designed around maximizing yield and product length while minimizing template-independent side reactions. Template-independent reactions compete with the desired template-dependent reaction, an undesirable situation that becomes more severe as less RNA template is used. Amplification products dominated by template-independent product result in greatly reduced sensitivity and compression of differences in microarray hybridization experiments. Most notably, the oligo(dT) primer used in reverse transcription (RT) yields a high-molecular-weight product in the in vitro transcription (IVT) reaction independent of any cDNA template. This protocol therefore limits the amount of primer used by using small cDNA synthesis volumes. In addition, high-molecular-weight, template-independent product is generated in the presence of biotinylated NTPs and the absence of any polymer when excessive amounts of T7 RNA polymerase activity are used. Template-dependent product of questionable molecular weight and limited functionality in downstream reactions can also be produced with excessive T7 RNA polymerase activity. Essentially, more yield is not always better. Here, a single round of amplification typically results in a fivefold to 20-fold mass conversion of starting material. If the first-round aRNA is used as a template for a second round of amplification, 200- to 400-fold amplification is typical.