Hybridization Efficiency Research Articles

Pollen biology and metaxenic studies on early maturing grape (Vitis vinifera) genotypes

The present experiment was conducted during 2020–21 and 2021–22 at ICAR-Indian Agricultural Research Institute, New Delhi to study the impact of open-, self-, and cross-pollination on metaxenic traits (berries) and the incidence of cleistogamy in grapes (Vitis vinifera L.). Seven different cross-combinations were used on the grape variety Pusa Navrang, which were planted at 3 m × 3 m spacing and trained on the Y-trellis training system. The experiment was laid out in a randomized block design (RBD) with four replications. The incidence of cleistogamy, berry set, retention percentage, and the metaxenia effect of pollen grains in different grapevine varieties, namely, Pusa Navrang (PN), Black Muscat (BM), Pusa Swarnika (PS), Beauty Seedless (BS), Perlette (PER), Male Hybrid (MH), were studied. Also, pollen quantity and viability were determined for the different grapevine varieties under study. Depending on the genotypes and whether the fruit set is obtained inside the covered flowers, cleistogamy was confirmed. A significantly higher berry set (40–85%) and retention (32–77%) were recorded in open- and self-pollinated flowers. The total number of pollens/anther in six grape genotypes ranged from 7916.62–23832.02, whereas the maximum active pollen rates were noted in the Male Hybrid (70.01%) and Black Muscat (66.80%). The pollens from the Beauty Seedless genotype exhibited significant enhancement of metaxenic traits such as berry set, retention, and size in Pusa Navrang. Therefore, utilizing Beauty Seedless as a pollen parent in grape breeding programmes holds promise for enhancing berry set and overall berry quality, thus enhancing the hybridization efficiency.

Challenges and solutions in FISH for formalin-fixed paraffin-embedded tissue: A scoping review.

Fluorescence in situ hybridization (FISH) has revolutionized molecular cytogenetic analysis since the 1980s, enabling precise localization of DNA sequences in cells and tissues. Despite its relevance, applying FISH to formalin-fixed paraffin-embedded (FFPE) tissue samples encounters significant technical challenges. This review addresses the main issues encountered in this context, such as inadequate fixation, contamination, block and slide age, inadequate pretreatment, and FISH technique. Proposed solutions include optimized pretreatment protocols, monitoring of blockage, careful selection of probes, and thorough analysis of results. Implementing good laboratory practices and quality control strategies are essential to ensure reliable results. Additionally, the use of emerging technologies such as artificial intelligence and digital pathology offers new perspectives for improving the efficiency and accuracy of FISH in FFPE samples. This review highlights the importance of a careful and personalized approach to overcome the technical challenges associated with FISH in FFPE samples, strengthening its role in research and clinical diagnosis. RESEARCH HIGHLIGHTS: Few FISH studies on FFPE: The scarcity of studies specifically addressing FISH applications in FFPE tissues highlights a critical gap in the literature. Troubleshooting FISH in FFPE tissues: Identifying and addressing common challenges in FISH techniques when applied to FFPE samples, such as signal quality and hybridization efficiency. Critical aspects of FISH technique: Discuss the main technical considerations crucial for successful FISH in FFPE tissues, including sample preparation, probe selection, and protocol optimization.

PCR-free and minute-scale electrochemical analysis of porcine DNA adulteration via molecularly amplified DNA sandwich assay

The increasing incidence of meat adulteration and mislabeling poses significant challenges in terms of food safety and consumer trust. This study proposes an electrochemical DNA biosensor for detecting porcine mitochondrial DNA in tainted meat products, offering a novel approach to address the above challenges. Unlike conventional nucleic acid amplification tests that rely on polymerase chain reactions (PCRs), the proposed biosensor employs a molecularly amplified DNA strategy with DNA tracers that bind to two regions of the target DNA, creating an elongated hybridization structure with multiple redox-tagging molecules. This design catalyzes detection signals autonomously, eliminating the need for PCR amplification. One-step DNA probe immobilization using poly-adenine (poly-A) oligonucleotides significantly improves hybridization efficiency and reduces the necessity for extensive sample purification, thereby simplifying the detection process. The proposed biosensor exhibits a linear detection range of 101–106 pM and a limit of detection (LOD) of 2.2 pM in controlled settings. Furthermore, the proposed biosensor distinguishes pork from beef in adulterated samples with a LOD of 1 % w/w. With its stability exceeding 9 weeks and a cost of less than 0.5 USD per test, the proposed biosensor offers a highly sensitive, economically viable solution with significant potential for widespread use in the meat industry and by end-users, effectively combating porcine adulteration.

Hybridization-driven synchronous regeneration of biosensing interfaces for Listeria monocytogenes based on recognition of fullerol to single- and double-stranded DNA

A novel sensitive and reusable electrochemical biosensor for Listeria monocytegenes DNA has been constructed based on the recognition of water-soluble hydroxylated fullerene (fullerol) to single- and double-stranded DNA. First, the fullerol was electrodeposited on glassy carbon electrode (GCE), acting as a matrix for non-covalent adsorption of single-stranded probe DNA. Upon hybridization with the target DNA, the double helix structure was formed and desorbed from the electrode surface, driving synchronous regeneration of the biosensing interfaces. The biosensor showed a probe DNA loading density of 144 pmol∙cm−2 with the hybridization efficiency of 72.2%. The biosensor is applicable for the analysis of target DNA in actual milk samples with recoveries between 101.0% and 104.0%. This sensing platform provides a simple method for the construction of sensitive and reusable biosensor to monitor Listeria monocytogenes-related food pollution.

HSP70 and APX1 play important roles in cotton male fertility by mediating ROS homeostasis

Male sterility is used in the production of hybrid seeds and can improve the breeding efficiency of cotton hybrids. Reactive oxygen species is closely associated with the tapetum and pollen development, but their relationship in cotton male fertility remains unclear. In this study, we comprehensively compared the cytology and proteome of the anthers from an Upland cotton (Gossypium hirsutum) material, Shida 98 (WT), and its nearly-isogenic male sterile line Shida 98A (MS). Cytology indicated delayed PCD in the tapetum and defects in microspores in MS anthers. And further studies revealed disruption of ROS homeostasis. Proteomic analysis identified proteins with differential abundance mainly being related to redox homeostasis, protein folding, and apoptotic signaling pathways. GhAPX1 interacted with GhHSP70 and played a crucial role in the development of cotton anthers. Exogenous application of HSP70 inhibitor increased H2O2 content and decreased the activity of APX1 and pollen viability. The GhAPX1 mutants generated by CRISPR/Cas9-mediated gene editing exhibited premature degradation of the tapetum, significant decrease in pollen viability, and significant increase in H2O2 content. Altogether, our results imply HSP70 and APX1 being the key players jointly regulating male fertility by mediating ROS homeostasis. These results provide insights into the proteins associated with male fertility.

Energy efficiency and electrochemical characteristic assessment of PEMFC-supercapacitor hybridization via railway profile

Electric trains equipped with efficient electric motors and powered by high-capacity hydrogen fuel cells are a choice of the global transportation industry to accelerate the global transition to a low-carbon transportation system. The operation of distinct vehicle types in varying geographical regions is characterized by unique features. Concurrently, the continually fluctuating demand for electricity necessitates the utilization of fuel cells in conjunction with energy storage devices. Thus, this study centers on examining the role of the PEMFC-supercapacitor direct hybridization system within the authentic driving profile of the subway in the Bangkok metropolitan region. The supercapacitor within the hybridization system serves as an energy management device, enhancing the durability of fuel cells when subjected to diverse load demands associated with train propulsion. In order to validate this hypothesis, an MRT (Mass Rapid Transit) protocol was formulated to emulate and serve as the load demand for the investigation, and then the PEMFC-supercapacitor direct hybridization system was operated using the created profile. Subsequently, the hybridization system undergoes electrochemical characterizations and fuel cell performance tests for diagnostic purposes. Empirical findings validated that the supercapacitor operates as a filter, generating a stable voltage response to varying load requirements, and possesses the capability to assimilate energy originating from regenerative braking. The integration of supercapacitors in stabilizing the operational parameters of proton exchange membrane fuel cells (PEMFCs) has markedly prolonged their operational lifespan. This assertion is substantiated by voltage signal analysis, which demonstrated a negligible increase in surface resistance of merely 0.23%, as depicted by Nyquist plots. Furthermore, cyclic voltammetry revealed that the system attained a peak electrochemical surface area (ECSA) of 0.0089 (106 cm2/mg of Pt). The information presented in this article constitutes data that has been minimally explored in existing research. Specifically, the study concentrates on the practical driving pattern of electric trains. Consequently, experimental results were deemed advantageous for the advancement of heavy-duty electric vehicles.

Fault detection and synchronization control in hybrid DC/AC microgrids using grid-forming inverter DC-link controller

This paper introduces a DC-link fault detection and synchronization control strategy for grid-forming inverters in hybrid DC/AC microgrids, aiming to bolster system stability and reliability. The proposed control scheme seamlessly integrates DC-link fault detection with virtual synchronous generator (VSG) control, facilitating inertia emulation and synchronization without the need for a phase-locked loop (PLL). The DC bus controller is designed to swiftly identify faults triggered by power imbalances, such as load fluctuations or generator integration, and dynamically adjust the power reference for the VSG controller to ensure inertia emulation. The Ziegler-Nichols method is utilized for meticulous tuning of the DC-link controller gains, while the modified swing equation governs VSG inertia and synchronization. MATLAB/Simulink demonstrate the superior performance of the proposed method compared to traditional GFM control strategies. Specifically, the proposed controller improved the frequency nadir to 59.98 Hz compared to 59.75 Hz and showed an enhanced power step response of 7.9×10^5 W compared to 6.9×10^5 W for conventional control method. The proposed GFM control also eliminated the 10×10^6 W switching spike observed with conventional controllers. These findings highlight the potential of this fault-controller approach to advance the reliability and efficiency of hybrid DC/AC microgrids.

Enhancing clinical genomic accuracy with panelGC: a novel metric and tool for quantifying and monitoring GC biases in hybridization capture panel sequencing.

Accurate assessment of fragment abundance within a genome is crucial in clinical genomics applications such as the analysis of copy number variation (CNV). However, this task is often hindered by biased coverage in regions with varying guanine-cytosine (GC) content. These biases are particularly exacerbated in hybridization capture sequencing due to GC effects on probe hybridization and polymerase chain reaction (PCR) amplification efficiency. Such GC content-associated variations can exert a negative impact on the fidelity of CNV calling within hybridization capture panels. In this report, we present panelGC, a novel metric, to quantify and monitor GC biases in hybridization capture sequencing data. We establish the efficacy of panelGC, demonstrating its proficiency in identifying and flagging potential procedural anomalies, even in situations where instrument and experimental monitoring data may not be readily accessible. Validation using real-world datasets demonstrates that panelGC enhances the quality control and reliability of hybridization capture panel sequencing.

Short- and long-term weather prediction based on a hybrid of CEEMDAN, LMD, and ANN.

Agriculture is one of the major economic sectors in Africa, and it predominantly depends on the climate. However, extreme climate changes do have a negative impact on agricultural production. The damage resulting from extreme climate change can be mitigated if farmers have access to accurate weather forecasts, which can enable them to make the necessary adjustments to their farming practices. To improve weather prediction amidst extreme climate change, we propose a novel prediction model based on a hybrid of complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN), local mean decomposition (LMD), and artificial neural networks (NN). A detailed comparison of the performance metrics for the short- and long-term prediction results with other prediction models reveals that the three-phase hybrid CEEMDAN-LMD-NN model is optimal in terms of the evaluation metrics used. The study's findings demonstrate the efficiency of the three-phase hybrid CEEMDAN-LMD-NN prediction model in decision-system design, particularly for large-scale commercial farmers, small-holder farmers, and the agricultural index insurance industry that require reliable forecasts generated at multi-step horizons.

A novel hybrid efficiency prediction model for pumping well system based on MDS–SSA–GNN

AbstractThe prediction of the efficiency of oil well pumping systems plays an important role in optimizing the energy efficiency parameters of these systems. Currently, the prediction of oil well pumping system efficiency relies primarily on mechanistic models, but these models are often overly complex in predicting efficiency. Some researchers have attempted to use deep learning to predict system efficiency, but due to insufficient consideration of influencing factors and the causal relationships between these factors and system efficiency, they often include irrelevant variables as influencing factors, leading to less accurate prediction models. In this paper, a hybrid model (MDS–SSA–GNN) is proposed for the prediction of pumping well system efficiency. The model consists of six parts: Pearson's product moment correlation coefficient (PPMCC), multidimensional scaling (MDS) transform, maximum–minimum normalization, sparrow optimization algorithm (SSA), graph neural network (GNN), and maximum–minimum inverse normalization. First, the size of the correlation coefficient between each influencing factor and the system efficiency is quantitatively calculated by using PPMCC. Second, the main influencing factors are downscaled by using MDS and normalized based on the principle of maximum–minimum normalization. Third, the GNN algorithm is used for the prediction of the pumping unit system efficiency, and the SSA algorithm is used for the optimization of the initial values of the network parameters. Finally, the prediction results are obtained by the maximum–minimum antinormalization. To validate the model's accuracy, this study randomly selected 100 actual oil wells for comparative analysis and analyzed the impact of structural parameters of the hybrid algorithm on the prediction accuracy of system efficiency. The analysis results demonstrate that the proposed model can effectively predict system efficiency and has a certain role in improving the accuracy of oil well pumping system efficiency predictions.

Interspecific Hardy Geranium Progenies: Morphological Characterization and Genetic Evaluation

Hardy geranium is a popular ornamental plant with a high market demand. As a result, there is a strong need for continuous innovation to improve its ornamental qualities. Interspecific hybridization is a widely used technique for introducing desirable traits and creating new cultivars with added value. To explore the possibilities of creating novelty within the Geranium genus, we conducted 2438 interspecific crosses over three flowering seasons, resulting in the successful harvest of 445 seeds and the final production of 82 seedlings. Using embryo rescue increased the germination rate of a seedling from 0.17% to 1.01%. To validate the paternal genetic contribution in the new seedlings, we used amplified fragment length polymorphism (AFLP) to assess the proportion of uniquely inherited paternal markers in all seedlings. AFLP analysis confirmed that 54 of the 82 seedlings were indeed true hybrids (65.8% hybridization success rate). A morphological evaluation of the flowers and leaves, including flower diameter (mm), style length (mm), number of flowers, flowering period (weeks), and color of both flowers and leaves, provided further confirmation of the F1 hybrid status of the seedlings. The results of our interspecific breeding demonstrated the efficiency of interspecific hybridization in hardy geraniums and its potential to create distinctive and novel cultivars for the ornamental market.

Sufficient energy band utilization profited from spatially discrete heterogeneous interfaces to induce efficient photoelectrochemical water splitting for ZnIn2S4 photoanode

The applied possibility of ZnIn2S4 is improved in the photoelectrochemical (PEC) energy transfer filed through elevated light absorption, accelerating charges transfer and suppressed carrier recombination. Consequently, Sb2S3/ZnIn2S4/Cu2S ternary composite photoanode is obtained through the induction of Sb2S3/ZnIn2S4 and ZnIn2S4/Cu2S heterogeneous interfaces at different spatial locations in ZnIn2S4, which enhances photocurrent density of ZnIn2S4-based photoanode to 2.81 mA/cm2 from 0.15 mA/cm2 with the nearly full visible spectrum absorption range of ∼ 776 nm. The deposition of Sb2S3 leads to enhanced quantum efficiencies, carrier diffusion time, and number of surface states. The loading of Cu2S top layer predominantly results in optimized surface states style and holes injection efficiency of SS/ZIS/CS. The comprehensive measurements and density functional theory (DFT) calculation demonstrate Sb2S3 and Cu2S are together employed to enhance the hybridization efficiencies of energy bands, accelerate charge transfer, revise the reaction sites, reduce free energy barrier of rate-determining steps and reaction over potential of ZnIn2S4 to enhance PEC water spitting. This work pioneers the construction of ZnIn2S4-based multijunction photoanodes with sufficient energy band utilization and confirms the conspicuous prospect for remaining photoanodes.

Amplification-free and label-free rapid detection of Staphylococcus aureus using solution-gated graphene transistor-based DNA biosensor with hybridization enhancement by interface engineering

Staphylococcus aureus (S. aureus) is one of the leading causes of foodborne illnesses worldwide, posing a significant risk to food quality and safety. However, conventional detection methods often require complex amplification and labeling procedures, which are time consuming and expensive. In this study, an extremely sensitive electrochemical biosensor using a solution-gated graphene transistor (SGGT) was constructed for the amplification-free and label-free rapid detection of S. aureus by utilizing specific single-stranded DNA (ssDNA) to modify the gold (Au) sensing gate. This modified ssDNA was designed to hybridize with the target S. aureus DNA sequence. Interface engineering of the ssDNA-modified sensing gate via optimization of specific target and probe sequences was proposed to improve its hybridization efficiency. This enabled the sensitive quantitative detection of S. aureus DNA. The resulting biosensor exhibited excellent specificity in distinguishing non-complementary, 3-base/9-base DNA mismatch targets of S. aureus DNA and the specific DNA sequences of other common pathogenic bacteria. It also enabled the rapid detection of extracted target DNA sequences in solution (within 27 min) and had a low limit of detection (LoD) of 10−17 M over a wide linear detection range (10−17–10−8 M). Furthermore, the newly-developed biosensor facilitated the real-time and on-site detection of the S. aureus genome (ATCC 6538) with LoD of 10−17 M (103 CFU/mL) in a portable smart-sensing system. This suggests a promising future for the development of rapid, label-free, and amplification-free detection of foodborne illnesses using low-cost, highly sensitive SGGT DNA biosensors with appropriate functionalization and interface engineering of the sensing electrode.

Identification, Elucidation and Deployment of a Cytoplasmic Male Sterility System for Hybrid Potato.

Recent advances in diploid F1 hybrid potato breeding rely on the production of inbred lines using the S-locus inhibitor (Sli) gene. As a result of this method, female parent lines are self-fertile and require emasculation before hybrid seed production. The resulting F1 hybrids are self-fertile as well and produce many undesirable berries in the field. Utilization of cytoplasmic male sterility would eliminate the need for emasculation, resulting in more efficient hybrid seed production and male sterile F1 hybrids. We observed plants that completely lacked anthers in an F2 population derived from an interspecific cross between diploid S. tuberosum and S. microdontum. We studied the antherless trait to determine its suitability for use in hybrid potato breeding. We mapped the causal locus to the short arm of Chromosome 6, developed KASP markers for the antherless (al) locus and introduced it into lines with T and A cytoplasm. We found that antherless type male sterility is not expressed in T and A cytoplasm, proving that it is a form of CMS. We hybridized male sterile al/al plants with P cytoplasm with pollen from al/al plants with T and A cytoplasm and we show that the resulting hybrids set significantly fewer berries in the field. Here, we show that the antherless CMS system can be readily deployed in diploid F1 hybrid potato breeding to improve hybridization efficiency and reduce berry set in the field.

A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

A study on the cutting efficiency of gas-laser hybrid cutting according to the optimal cutting speed when cutting 100 mm ultra-thick plates

A study on the cutting efficiency of gas-laser hybrid cutting according to the optimal cutting speed when cutting 100 mm ultra-thick plates

Engineering a Rigid Nucleic Acid Structure to Improve the Limit of Detection for Genetic Assays.

Detecting nucleic acids at ultralow concentrations is critical for research and clinical applications. Particle-based assays are commonly used to detect nucleic acids. However, DNA hybridization on particle surfaces is inefficient due to the instability of tethered sequences, which negatively influences the assay's detection sensitivity. Here, we report a method to stabilize sequences on particle surfaces using a double-stranded linker at the 5' end of the tethered sequence. We termed this method Rigid Double Stranded Genomic Linkers for Improved DNA Analysis (RIGID-DNA). Our method led to a 3- and 100-fold improvement of the assays' clinical and analytical sensitivity, respectively. Our approach can enhance the hybridization efficiency of particle-based assays without altering existing assay workflows. This approach can be adapted to other platforms and surfaces to enhance the detection sensitivity.

Multiarmed DNA jumper and metal-organic frameworks–functionalized paper-based bioplatform for small extracellular vesicle–derived miRNAs assay

Small extracellular vesicle–derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF–rGO–Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.

PNA-Functionalized, Silica Nanowires-Filled Glass Microtube for Ultrasensitive and Label-Free Detection of miRNA-21.

MicroRNAs (miRNAs) are endogenous and noncoding single-stranded RNA molecules with a length of approximately 18-25 nucleotides, which play an undeniable role in early cancer screening. Therefore, it is very important to develop an ultrasensitive and highly specific method for detecting miRNAs. Here, we present a bottom-up assembly approach for modifying glass microtubes with silica nanowires (SiNWs) and develop a label-free sensing platform for miRNA-21 detection. The three-dimensional (3D) networks formed by SiNWs make them abundant and highly accessible sites for binding with peptide nucleic acid (PNA). As a receptor, PNA has no phosphate groups and exhibits an overall electrically neutral state, resulting in a relatively small repulsion between PNA and RNA, which can improve the hybridization efficiency. The SiNWs-filled glass microtube (SiNWs@GMT) sensor enables ultrasensitive, label-free detection of miRNA-21 with a detection limit as low as 1 aM at a detection range of 1 aM-100 nM. Noteworthy, the sensor can still detect miRNA-21 in the range of 102-108 fM in complex solutions containing 1000-fold homologous interference of miRNAs. The high anti-interference performance of the sensor enables it to specifically recognize target miRNA-21 in the presence of other miRNAs and distinguish 1-, 3-mismatch nucleotide sequences. Significantly, the sensor platform is able to detect miRNA-21 in the lysate of breast cancer cell lines (e.g., MCF-7 cells and MDA-MB-231 cells), indicating that it has good potential in the screening of early breast cancers.

Laser Scribing for Perovskite Solar Modules of Long‐Term Stability

Although the efficiency of hybrid lead‐halide perovskite solar cells has been significantly improved, the efficiency gap between small‐area cells and large modules continues to be a considerable challenge. Laser scribing is essential for realizing high‐quality monolithic connections; however, the laser‐induced material changes and their correlation with device performance have not been yet well understood, in particular for the perovskite material systems. In this study, the effect of P3 laser processing conditions on device performance and stability is explained. The most interesting finding is an improvement in open‐circuit voltage (VOC) after aging and long‐term stability under low‐laser‐overlap conditions that avoid direct laser exposure to perovskite material system as a source of material degradation. It is found that a high‐laser‐overlap during P3 results in a lower fill factor after aging and accelerated degradation due to larger portion of perovskite directly exposed to laser during the scribing process. The increased VOC under low‐overlap conditions is attributed to the increased PbI2 formation in the P3 region. Moreover, a minimal pulse overlap is favorable for preserving long‐term device stability. Finally, a perovskite minimodule with an efficiency of 20.24% is successfully developed as a result of these findings.