Amplification Cycles Research Articles

A Programmable Bio-Chip With Adaptive Pattern-Control Micro-Electrode-Dot-Array

A digital lab-on-CMOS chip is presented for nucleic acid amplification test. This biochip consists of programmable microelectrode dot array and digital timing controller. With novel circuit design in each microelectrode, microfluidic operations with velocity of 0.6mm/s, temperature profile with maximum change rate of 4.5°C/s and location sensing in <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu $ </tex-math></inline-formula> second-level can be achieved. As a result, test samples can be located first, followed by target operations to meet the requirements of specific medical tests with less reagent consumption and test time/cost. Furthermore bio-protocols can be derived to ensure both replicability and reliability in various medical tests. Preliminary results in loop-mediated isothermal amplification (LAMP) and PCR thermal cycle tests demonstrate the feasibility of our proposal with less test time of < 40 min and < 1 min respectively, making our proposal very suitable for mobile healthcare applications.

Toehold-mediated strand displacement reaction-propelled cascade DNAzyme amplifier for microRNA let-7a detection

DNAzyme amplifiers have been extensively explored as a useful sensing platform, but single DNAzyme amplifier is limited in biosensing applications by its low sensitivity. Herein, a cascade DNAzyme amplifier was designed by exploiting concurrent amplification cycle principles of toehold-mediated strand displacement reaction (TSDR) and Zn2+-assisted DNAzyme cycle with lower cost and simpler procedures. Compared with single DNAzyme amplifier, the proposed TSDR-propelled cascade DNAzyme amplifier exhibited higher sensitivity by releasing more DNAzyme through TSDR to cleave substrate strand during the DNAzyme cycle. Base on this, let-7a could be sensitively detected in the range of 5–50 nmol/L with a detection limit of 64 pmol/L. Furthermore, the dual signal amplification strategy of the cascade DNAzyme amplifier exhibited excellent selectivity to distinguish single-base mismatched DNA strands, which has been successfully applied to the determination of let-7a in blood serum, showing high promise in early cancer diagnosis.

Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer.

BACKGROUNDGastric cancer (GC) is one of the most prevalent malignant tumors that endangers human health. Early diagnosis is essential for improving the prognosis and survival rate of GC patients. Ring finger protein 180 (RNF180) is involved in the regulation of cell differentiation, proliferation, apoptosis, and tumorigenesis, and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC. Thus, methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIMTo use droplet digital polymerase chain reaction (ddPCR) to quantify the methylation level of the RN180 gene. A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODSThe primer and probe were designed and selected, the conversion time of bisulfite was optimized, the ddPCR system was adjusted by primer concentration, amplification temperature and amplification cycles, and the detection limit of ddPCR was determined.RESULTSThe best conversion time for blood DNA was 2 h 10 min, and that for plasma DNA was 2 h 10 min and 2 h 30 min. The results of ddPCR were better when the amplification temperature was 56 °C and the number of amplification cycles was 50. Primer concentrations showed little effect on the assay outcome. Therefore, the primer concentration could be adjusted according to the reaction system and DNA input. The assay required at least 0.1 ng of input DNA.CONCLUSIONIn summary, a ddPCR assay was established to detect methylated RNF180, which is expected to be a new diagnostic biomarker for GC.

Comparison of antigen test and polymerase chain reaction for SARS-CoV-2 in children younger than 12 years.

Stopping the spread of coronavirus disease 2019 (COVID-19) is critical and can be achieved through rapid and effective detection techniques. Our objective was to compare the diagnostic accuracy of rapid antigen tests (RAgT) and reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) and to describe amplification cycle thresholds (Cts). Participants were children aged 1 month to 11 years with symptoms for less than 7 days, who did not have a detectable result in the past 90 days, and were immunocompetent. A total of 1855 patients were included; the prevalence of COVID-19 was 4.7%. For the RAgT, overall sensitivity was 60.2% and specificity, 99.8%; in children older than 5 years, values were 69.8% and 99.8%, respectively. Ct values for discordant samples were higher. To conclude, the diagnostic accuracy indicated that the specificity of RAgT is similar to that of RT-qPCR, but its sensitivity is notably lower, especially in children younger than 5 years.

Highly sensitive single-cell chromatin accessibility assay and transcriptome coassay with METATAC

Recent advances in single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) and its coassays have transformed the field of single-cell epigenomics and transcriptomics. However, the low detection efficiency of current methods has limited our understanding of the true complexity of chromatin accessibility and its relationship with gene expression in single cells. Here, we report a high-sensitivity scATAC-seq method, termed multiplexed end-tagging amplification of transposase accessible chromatin (METATAC), which detects a large number of accessible sites per cell and is compatible with automation. Our high detectability and statistical framework allowed precise linking of enhancers to promoters without merging single cells. We systematically investigated allele-specific accessibility in the mouse cerebral cortex, revealing allele-specific accessibility of promotors of certain imprinted genes but biallelic accessibility of their enhancers. Finally, we combined METATAC with our high-sensitivity single-cell RNA sequencing (scRNA-seq) method, multiple annealing and looping based amplification cycles for digital transcriptomics (MALBAC-DT), to develop a joint ATAC-RNA assay, termed METATAC and MALBAC-DT coassay by sequencing (M2C-seq). M2C-seq achieved significant improvements for both ATAC and RNA compared with previous methods, with consistent performance across cell lines and early mouse embryos.

G-quadruplex-deficient precursor hairpin probes for ultra-low background dual-mode detection of miRNAs

Design of oligonucleotide probe-based isothermal amplification with the ability to identify miRNA biomarkers is crucial for molecular diagnostics. Herein, we engineered a miRNA-21 responsive G-quadruplex-deficient precursor hairpin probe (PHP) to achieve dual-mode detection of fluorescent signal and colorimetric signal. Due to lack of complete G-quadruplex sequence, PHP becomes shorter in length, lower background signal and less interference. Based on the polymerase-driven amplification mechanism, in the presence of miRNAs, two simultaneous amplification reaction processes will occur in PHP: miRNA-based amplification process and endogenous amplification process along the 3′ end. Due to the positional difference between the starting points of the two amplification processes, the orderly and efficient occurrence of the two amplification processes can be achieved. Based on an interesting concept, PHP can achieve high detection performance with only simple amplification cycles. In such a way, the detection limits for fluorescence and colorimetry were 2.93 fM and 8.81 fM, which would cover most of clinical qualitative and quantitative needs. Thus, the accurate quantitative and visual miRNA detection technology based on PHP is beneficial to carry out extensive disease screening and treatment monitoring in various complex occasions.

Quantitative detection of Vibrio parahaemolyticus in aquatic products by duplex droplet digital PCR combined with propidium monoazide

Based on the combination of droplet digital PCR (ddPCR) and propidium monoazide (PMA), a new method called PMA-duplex ddPCR was developed for the quantitative determination of viable Vibrio parahaemolyticus in aquatic products. Specific primers and probes targeting a new gene, vp0488, coding for putative DNA polymerase III beta chain, which was self-screened by this laboratory, and a traditional target gene, thermolabile hemolysin (tlh), were designed and verified. The duplex ddPCR system and conditions and PMA treatment conditions were optimized. The limit of detection (LOD) for PMA-duplex ddPCR was evaluated and applied to detect V. parahaemolyticus in artificially contaminated samples and actual samples. The results indicate that the optimal duplex ddPCR annealing temperature was 60 °C, the optimal number of amplification cycles was 40, and the optimal ratio of primer to probe was 1:0.5. The optimal PMA treatment conditions were as follows: incubation time 5 min, concentration 30 μmol/L, and exposure time 15 min. The LOD of PMA-duplex ddPCR (7.32 × 101 CFU/mL) was lower than that of duplex quantitative PCR (qPCR) (7.32 × 102 CFU/mL). The lowest detectable V. parahaemolyticus in oysters was 8.15 × 101 CFU/g without any pre-enrichment. The method established in this study has the advantages of good specificity, high sensitivity and accuracy, which provides an effective method for the detection of viable Vibrio parahaemolyticus in aquatic foods.

전달경로분석법을 이용한 상용 전기 차량의 크립 그론 소음 저감 연구

Creep groan noise is a low-frequency, structure-borne noise that generally occurs when a vehicle starts moving from a standstill or comes to a gradual stop with minimal brake operation. It is generated by friction-induced vibrations from the stick-slip cycle of the brakes and are typically transmitted through the suspension. The investigations in this paper aim to reduce the creep groan noise by considering the frequency characteristics of the suspension. Transfer path analysis (TPA) is performed to identify the noise contribution of each suspension component. Thereafter, the cause of acoustic amplification and stick-slip cycle of creep groan are investigated through modal testing on the primary transfer path components. The noise level and trigger rate of creep groan noise are reduced by suppressing the behavior of the components using a dynamic vibration absorber (DVA). This shows the possibility of creep groan noise reduction from the perspective of the vehicle’s transfer system.

Ratiometric Fluorescent Biosensor Based on Self-Assembled Fluorescent Gold Nanoparticles and Duplex-Specific Nuclease-Assisted Signal Amplification for Sensitive Detection of Exosomal miRNA.

The sensitive detection of cancer-associated exosomal microRNAs shows enormous potential in cancer diagnosis. Herein, a ratiometric fluorescent biosensor based on self-assembled fluorescent gold nanoparticles (Au NPs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for sensitive detection of colorectal cancer (CRC)-associated exosomal miR-92a-3p. In this biosensing system, the hairpin DNA modified with sulfhydryl and fluorescent dye Atto-425 at both ends is conjugated to fluorescent Au NPs through Au-S bonds, resulting in the quenching of Atto-425. The miR-92a-3p can open the hairpin of DNA and forms an miR-92a-3p/DNA heteroduplex, triggering the specific cleavage of DSN for the DNA in the heteroduplex. As a result, Atto-425 leaves the fluorescent Au NPs and recovers the fluorescence emission. The released miR-92a-3p can hybridize with another hairpin DNA and lead to a stronger fluorescence recovery of Atto-425 to form a signal amplification cycle. The stable fluorescence of Au NPs and the changing fluorescence of Atto-425 constitute a ratiometric fluorescent system reflecting the concentration of miR-92a-3p. This biosensor exhibits excellent specificity and can distinguish CRC patients from healthy individuals by detecting miR-92a-3p extracted from clinical exosome samples, showing the potential in CRC diagnosis.

Ion Torrent ™ Genexus ™ Integrated Sequencer and ForeNGS Analysis Software—An automatic NGS-STR workflow from DNA to profile for forensic science

The Ion Torrent ™ Genexus ™ Sequencer (Genexus) is a highly integrated instrument that can automate library construction, templating, and sequencing in a single-instrument run. By programing the ForeNGS Analysis Software (FNAS), we bridged the gap between sequencing and genotyping without manual intervention. FNAS can automatically transfer sequencing output files from Genexus, analyze the repeat and flanking regions aligned to the GRCh38 assembly, name the alleles according to the ISFG guidelines, and generate user-friendly interactive profiles. Genexus and FNAS can accomplish the fully automatic DNA-to-Profile workflow in forensics. Based on our experiences, the optimal assay parameters on Genexus were validated as follows: 24 cycles of target amplification for library construction; 40 μL of library and 400 bp of template size for templating; 852 flows of dNTPs by order of Ion samba HID2 for sequencing; and 750,000 reads per sample at minimum for 16 samples multiplexed on a lane. By developmental validations of the Precision ID Globalfiler ™ NGS STR Panel v2, Genexus presented competitive performance at the optimal assay parameters qualified to detect commonly used forensic STR markers. It could produce repeatable and reproducible results, and human profiles could be easily separated from nonhuman profiles. Additionally, Genexus was sensitive enough to detect samples with 100 pg of input DNA, and it was suitable for various types of case samples, especially for low copy number samples and degraded samples. Moreover, minor contributors could be detected between the 4:1 and 1:4 mixtures with an analysis threshold of 50 × . The Genexus workflow is a robust and labor-effective solution enabling forensic scientists to obtain NGS-STR profiles within a single day and with only the need to prepare DNA extracts, then set up Genexus, and finally interpret profiles on FNAS.

629 Performance monitoring of a streamlined and scalable non-invasive gene expression assay for pigmented lesions

The DermTech Pigmented Lesion Assay (PLA) is a qPCR-based assay that aids in the detection of melanoma by assessing up-regulation of two melanoma-associated genes, LINC00518 and PRAME, in skin samples collected by non-invasive adhesive patches. This study is to establish assay performance characteristics of the PLA post optimization, and to investigate the prognostic potential of the assay by comparison of qPCR amplification cycle in PLA positive samples with the histopathologic diagnoses of cutaneous pigmented lesions. RNA isolated from non-invasively collected skin cells was analyzed from seventy-five pigmented lesions. The PLA was optimized to simplify the laboratory workflow and increase scalability of the assay through a series of process improvements including RNA quantification by 1-step qRT-PCR (vs. reverse transcription followed by qPCR) and assay miniaturization. PLA result was determined by evaluating qPCR amplification of LINC00518 and PRAME multiplexed with housekeeping genes β-Actin and PPIA respectively. Samples that demonstrated over-expression of LINC00518 and PRAME relative to the housekeeping genes were considered PLA positive. Blinded to the PLA results, biopsy specimens from the pigmented lesions were examined and classified by a dermatopathologist as invasive melanoma (n=22); melanoma in situ (MIS) (n=26); benign nevus (24); or other (n=3). All twenty-two lesions classified as invasive melanoma by histopathology were PLA-positive, with an average LINC00518 Ct of 32.2 and PRAME Ct of 30.9. Seventeen of the twenty-six samples classified as MIS were PLA-positive, with an average LINC00518 Ct of 33.8 and PRAME Ct of 33.2. Of the twenty-seven samples not classified as melanoma, five were PLA-positive, with an average LINC00518 Ct of 34.4 and PRAME Ct of 35.8. The results indicate that PLA Ct values are highly correlated with histopathologic diagnoses. The optimized PLA assay demonstrated a sensitivity of 100% for invasive melanoma and 65% for MIS, with an overall specificity of 81%.

Ratiometric Fluorescence Detection of Colorectal Cancer-Associated Exosomal miR-92a-3p with DSN-Assisted Signal Amplification by a MWCNTs@Au NCs Nanoplatform.

The detection of miRNA shows great promise in disease diagnosis. In this work, a ratiometric fluorescent biosensor based on multi-walled carbon nanotubes@gold nanoclusters (MWCNTs@Au NCs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for miRNA detection. Colorectal cancer (CRC)-associated miR-92a-3p extracted from exosomes was selected as the target. MWCNTs@Au NCs performs the dual functions of fluorescence quencher and internal fluorescence reference. In the absence of miR-92a-3p, an Atto-425-modified single-stranded DNA probe is adsorbed on MWCNTs@Au NCs, resulting in the quenching of Atto-425. In the presence of miR-92a-3p, the duplex is formed by hybridization of the probe and miR-92a-3p and leaves the MWCNTs@Au NCs, resulting in the fluorescence recovery of Atto-425. DSN can cleave the probe and result in the release of miR-92a-3p. The released miR-92a-3p can hybridize with other probes to form a signal amplification cycle. The fluorescence of MWCNTs@Au NCs remains stable and constitutes a ratiometric fluorescence system with that of Atto-425. A detection concentration interval of 0.1–10 pM and a limit of detection of 31 fM was obtained under optimized measurement conditions. In addition, the accuracy of the biosensor was validated by detecting the concentration of miR-92a-3p extracted from clinical exosome samples.

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.

Detection of Newcastle disease virus and H5-subtype Influenza virus in swiftlet houses by Multiplex reverse transcription PCR assay

The purpose of this study was to investigate the presence of Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), and Avian influenza type A, especially H5 subtype (AIV-H5) in swiftlet houses and swiftlet nest by multiplex RT-PCR (mRT-PCR). The assay used two specific primer pairs designed to detect the conserved sequence of the F gene of NDV and the HA gene of the AIV-H5, with product sizes of 282 bp and 420 bp, respectively. The mRT-PCR was established with detection limit of 25 copies/reaction for each target virus. The thermal cycle was optimized as follows: cDNA synthesis at 45ºC for 20 min, an initial denaturation at 95ºC for 5 min, followed by 35 cycles of amplification encompassing denaturation at 95ºC for 30 sec, annealing at 58ºC for 30 sec, and extension at 72ºC for 45 sec, ending by a final extension step at 72ºC for 7 min. Eighty-eight field samples including feces and swabs of the nest surface were examined and all samples were confirmed to be negative for these two viruses. The results of this study indicated that the swiftlet nests and the environment of swiftlet houses were not contaminated with NDV or AIV-H5 viruses. Moreover, the established mRT-PCR protocol had good specificity, detection limit, and can apply for routine veterinary diagnosis.

PB1791: ON THE ISSUE OF HOUSEKEEPING GENE PRIMER DESIGN FOR ADEQUATE ASSESSMENT OF THE EXPRESSION OF THE CHIMERIC TRANSCRIPTS.

Background: Chimeric transcripts, which are the markers of hematological malignancies, is often assessed by expression level relative to the control housekeeping gene. The critical step for the analysis is RNA isolation and subsequent cDNA synthesis. Routine RNA isolation methods do not guarantee the absence of genomic DNA impurities, which may interfere with cDNA amplification. This results in the bias in control gene expression and underestimation of the relative expression. The use of sophisticated RNA isolation procedures or treatment with RNase-free DNAses significantly complicates and increases the cost of analysis, and can also lead to degradation of target sequences by endogenous nucleases. Here we describe a simple and effective solution to this problem. Aims: To optimize the RT-PCR approach for quantitative assessment of the chimeric transcripts gene expression relative to the control ABL1 gene, for the insensitivity to genomic DNA impurities. Methods: Total RNA was isolated from peripheral blood leukocytes or bone marrow cells as described by Chomczynski (1987). RT-PCR was performed using reagents and primers from Synthol (Moscow, Russia). Results: For ABL1 mRNA amplification primers flanking 2 gene introns were designed (see Table 1). This precludes PCR amplification of the corresponding (139708bp long) genomic sequence. At the same time, the cDNA of this fragment is only 100 nucleotides long. The primers recommended by Europe Against Cancer Association (EAC) and widely used (including as part of commercial diagnostic kits) flank 687bp ABL1 gene DNA fragment. In this case, the admixture of genomic DNA is co-amplified with the cDNA transcript in PCR. We have compared CBFB::MYH11 chimeric transcript relative expression levels in patients with AML, obtained with ABL-EAC primers and those proposed by us (ABL-N). We have tested 40 cDNA samples obtained from diverse RNA isolation routines, including 8 archival samples stored in the freezer for more than 6 months. The threshold cycles of amplification (ΔCt) for the samples from freshly isolated RNA were mostly the same with ABL-EAC and ABL-N primers. For archival RNA, cDNA, and RNA isolated from frozen cells, ΔCt with ABL-N primers drops significantly while ΔCt with ABL-EAC primers is preserved. This may indicate degradation in archival RNA and cDNA samples, while the level of genomic DNA decreases slightly. Thus, usage of ABL-EAC primers may lead in some circumstances to a significant bias in chimeric transcript evaluation. While primers we offer (ABL-N) are still effective even in the cases with compromised sample quality. Image:Summary/Conclusion: Genomic DNA amplification does not tamper with fusion gene transcripts RT-PCR measurement due to the absence of an appropriate target in the genomic DNA. While primers for measuring housekeeping genes expression (even those widely used) may have these targets and therefore amplify genomic DNA along with cDNA. Here we propose primers for measuring ABL1 control gene expression allowing reliable calculation of chimeric transcripts relative expression, regardless of RNA sample purity, in contrast to primers recommended by the EAC.

Validation of Non-Invasive Preimplantation Genetic Screening Using a Routine IVF Laboratory Workflow.

Embryo selection is needed to optimize the chances of pregnancy in assisted reproduction technology. This study aimed to validate non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using a routine IVF laboratory workflow. Can niPGT-A combined with time-lapse morphokinetics provide a better embryo-selection strategy? A total of 118 spent culture mediums (SCMs) from 32 couples were collected. A total of 40 SCMs and 40 corresponding trophectoderm (TE) biopsy samples (n = 29) or arrested embryos (n = 11) were assessed for concordance. All embryos were cultured to the blastocyst stage (day 5 or 6) in a single-embryo culture time-lapse incubator. The modified multiple annealing and looping-based amplification cycle (MALBAC) single-cell whole genome amplification method was used to amplify cell-free DNA (cfDNA) from the SCM, which was then sequenced on the Illumina MiSeq system. The majority of insemination methods were conventional IVF. Low cfDNA concentrations were noted in this study. The amplification niPGT-A and conventional PGT-A was 67.7%. Based on this study, performing niPGT-A without altering the daily laboratory procedures cannot provide a precise diagnosis. However, niPGT-A can be applied in clinical IVF, enabling the addition of blastocysts with a better prediction of euploidy for transfer.

Karyotype of the Blastocoel Fluid Derived by Laser-Assisted Hatching Demonstrates a Low Agreement With the Trophectoderm

ObjectiveThe aim of this study is to compare the amplification efficiency and the genomic profiles of blastocoel fluid (BF) derived by laser-assisted hatching and trophectoderm (TE) cells derived from the same blastocyst.MethodsFifty-four fresh blastocysts underwent shrinkage by laser-assisted hatching, and each BF sample was collected individually. BF and TE cells were retrieved from each blastocyst for chromosome analysis through multiple annealing and looping-based amplification cycles (MALBAC) and next-generation sequencing (NGS).ResultsFifty-four BF samples and 32 TE samples were retrieved for this study. Out of the 54 BF samples, only 35 provided reliable NGS data for comprehensive chromosome analysis (64.8%), while all 32 TE samples did (100%). Finally, there were 23 pairs of BF and TE samples from the same blastocyst. Only 17.4% of the BF-DNA karyotypes were completely agreeable with the TE samples (4/23).ConclusionBlastocoel fluid derived by laser-assisted hatching is easy to operate, and BF-DNA can be successfully amplified and subjected to NGS. Due to the low amplification efficiency and increased discordance with TE, BF does not adequately represent the status of the rest of the blastocyst. The use of BF as a single source of DNA for preimplantation genetic screening (PGS) is not yet advised.

Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow

AimTo evaluate critical steps in Illumina® Human mtDNA Genome assay: target enrichment, limited-cycle PCR, and library normalization, in order to optimize the protocol for analysis of whole mitochondrial genomes from human reference samples.MethodsThree long-range high-fidelity DNA polymerases (PlatinumTM PCR SuperMix High Fidelity, LA Taq® Hot Start, and PrimeSTAR® GXL) were tested for their performance in the amplification of mtDNA fragments. Sequencing results of ten samples, as well as negative controls, which underwent library preparation with 12 and 15 cycles in limited-cycle PCR were compared. Additionally, two library normalization methods were compared: bead-based normalization vs quantification and individual normalization.ResultsPrimeSTAR® GXL performed best for mitochondrial DNA enrichment. Increment of amplification cycles to 15 in limited-cycle PCR step did not affect either the sequencing process or variant calling. Library quantification combined with individual library-by-library dilution outperformed bead-based normalization.ConclusionOptimizations described herein provide beneficial insights for laboratories aiming at implementation and/or advancement of similar massively parallel sequencing workflows (eg, small genomes, PCR amplicons, and plasmids).

Stochastic expression of invasion genes in Plasmodium falciparum schizonts

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms. Here, we investigate transcriptional heterogeneity and stochastic gene expression in Plasmodium falciparum by performing the quasilinear multiple annealing and looping based amplification cycles (MALBAC) based amplification and single cell RNA sequencing of blood stage schizonts. Our data reveals significant transcriptional variations in the schizont stage with a distinct group of highly variable invasion gene transcripts being identified. Moreover, the data reflects several diversification processes including putative developmental “checkpoint”; transcriptomically distinct parasite sub-populations and transcriptional switches in variable gene families (var, rifin, phist). Most of these features of transcriptional variability are preserved in isogenic parasite cell populations (albeit with a lesser amplitude) suggesting a role of epigenetic factors in cell-to-cell transcriptional variations in human malaria parasites. Lastly, we apply quantitative RT-PCR and RNA-FISH approach and confirm stochastic expression of key invasion genes, such as, msp1, msp3, msp7, eba181 and ama1 which represent prime candidates for invasion-blocking vaccines.

High prevalence of Ichthyophonus sp. infections in Northeast Atlantic mackerel (Scomber scombrus).

High prevalence of Ichthyophonus sp. infections in Northeast Atlantic mackerel (Scomber scombrus).