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PubMed2025

RNA Sequencing Protocols for Short-Read Sequencing.

RNA sequencing (RNA-seq) methodologies allow the discovery of novel variants and transcripts. These comprise three general steps: (1) capture of RNA species of interest, (2) conversion of RNA to complementary DNA (cDNA), and (3) modification of cDNA to fit the sequencing platform. Here we describe four different library preparation protocols for short-read sequencing: cDNA synthesis with poly(A) selection, library preparation with ribosomal depletion, and cDNA synthesis with SMART® (Switching Mechanism at 5' end of RNA Template) technology for low and Pico inputs.

Gene Library↗

PubMed2026

Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the lagging strand. In addition to primers, RDHs form through misincorporation of ribonucleotides by DNA polymerase during elongation and by formation of R loops during transcription. R loops are three-stranded structures that form when the nascent mRNA anneals to the template DNA strand, displacing the complementary DNA strand. The persistence of RDHs is deleterious to genome stability in all cells because they increase susceptibility to mutations, impaired replication fork progression, DNA double-stranded breaks, and genomic rearrangements. In many bacteria, it is well established that components of the replicative DNA polymerase form a macromolecular complex that can be imaged using single-molecule or ensemble fluorescence approaches. The spatiotemporal regulation of proteins involved in RDH removal during lagging-strand maturation is less clear. Here, we study three proteins that are involved in the removal of RDHs from the lagging strand during DNA replication in the Gram-positive bacterium Bacillus subtilis: DNA polymerase I (Pol I), FenA, and RNase HIII. We characterized the behavior of each PAmCherry-tagged lagging-strand enzyme in living cells using single-particle tracking photoactivated localization microscopy. We find that all three proteins are highly mobile, suggesting residence times at their target substrates are below our temporal resolution. We also find evidence that Pol I activity is modulated through interaction with the replisome, whereas FenA and RNase HIII are regulated through access to the nucleoid. Our results provide new insight into how enzymes are recruited to resolve RDHs during lagging-strand replication in vivo.

DNA Replication↗

PubMed2010

Gene therapy of mdx mice with large truncated dystrophins generated by recombination using rAAV6.

Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer represents a promising approach for many diseases. However, the applicability of rAAV vectors has long been hindered by the small (~4.8 kb) DNA packaging capacity. This limitation can hamper the packaging and delivery of critical regulatory elements and/or larger coding sequences, such as the ~14-kb dystrophin complementary DNA (cDNA) that is of interest for gene therapy of Duchenne muscular dystrophy (DMD). Here, we have demonstrated reconstitution of an expression cassette (7.3 kb) encoding a highly functional "minidystrophin" protein (ΔH2-R19, 222 kd) in vivo following intravascular co-delivery of two independent rAAV6 vectors sharing a central homologous recombinogenic region of 372 nucleotides. Similar to previously reported trans-splicing approaches, one rAAV vector provides the promoter with the ~1/2 initial portion of minidystrophin, while the second vector provides the remaining minidystrophin cDNA followed by the polyadenylation signal. Significantly, administering a modest dose [2 × 10(12) vector genomes (vg)] of the two minidystrophin-encoding rAAV vectors to dystrophic mice elicited an improvement of physiological performance indicative of prevention or amelioration of the disease state. These studies provide evidence that functional dystrophin transgenes larger than that typically carried by a single rAAV genome can be reconstituted in vivo by homologous recombination (HR) following intravascular co-delivery with rAAV6.

Animals↗

PubMed2024

Regulation of DNA Topology in Archaea: State of the Art and Perspectives.

DNA topology is a direct consequence of the double helical nature of DNA and is defined by how the two complementary DNA strands are intertwined. Virtually every reaction involving DNA is influenced by DNA topology or has topological effects. It is therefore of fundamental importance to understand how this phenomenon is controlled in living cells. DNA topoisomerases are the key actors dedicated to the regulation of DNA topology in cells from all domains of life. While significant progress has been made in the last two decades in understanding how these enzymes operate in vivo in Bacteria and Eukaryotes, studies in Archaea have been lagging behind. This review article aims to summarize what is currently known about DNA topology regulation by DNA topoisomerases in main archaeal model organisms. These model archaea exhibit markedly different lifestyles, genome organization and topoisomerase content, thus highlighting the diversity and the complexity of DNA topology regulation mechanisms and their evolution in this domain of life. The recent development of functional genomic assays supported by next-generation sequencing now allows to delve deeper into this timely and exciting, yet still understudied topic.

Archaea↗

PubMed2026

Dogme: a nextflow pipeline for reprocessing nanopore RNA and DNA modifications.

MOTIVATION: Oxford Nanopore (ONT) sequencing allows for the direct detection of RNA and DNA modifications from unamplified nucleic acids, which is a significant advantage over other platforms. However, the rapid updates to ONT basecalling models and the evolving landscape of computational tools for modification detection bring about challenges for reproducible and standardized analyses. To address these challenges, we developed Dogme to automate basecalling, alignment, modification detection, and transcript quantification. Dogme automates the reprocessing of ONT POD5 files by integrating basecalling using Dorado, read mapping using minimap2 and subsequent analysis steps such as running modkit. The pipeline supports three major types of sequencing data-direct RNA (dRNA), complementary DNA (cDNA), and genomic DNA (gDNA). Dogme facilitates detection of diverse RNA modifications supported by Dorado such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, pseudouridine, 2'-O-methylation (Nm) and DNA methylation, while concurrently quantifying full-length transcript isoforms LR-Kallisto for transcript quantification for dRNA and cDNA. RESULTS: We applied Dogme to three separate mouse C2C12 myoblast replicates using direct RNA sequencing on MinION flow cells. We detected 96 603 m6A, 43 476 m5C, 8829 inosine, 10 055 pseudouridine, and 30 320 Nm sites in three biological replicates. The pipeline produced reproducible modification profiles and transcript expression levels across replicates, demonstrating its utility for integrative long-read transcriptomic and epigenomic analyses. AVAILABILITY AND IMPLEMENTATION: Dogme is implemented in Nextflow and is freely available under the MIT license at https://github.com/mortazavilab/dogme, with documentation provided for installation and usage.

RNA↗

PubMed2026

XXYLT1 and Mendelian Retinal Dystrophy.

IMPORTANCE: Substantial unexplained heritability remains for pathogenic inherited retinal disease (IRD) variants. Application of genome-wide association studies (GWAS) could help identify causal genes in rare diseases. OBJECTIVE: To leverage a GWAS for the discovery of IRD-associated genes. DESIGN, SETTING, AND PARTICIPANTS: This GWAS analysis was combined with replication of findings in 2 independent IRD cohorts. The study was conducted from January 2024 to December 2025 in a multicenter setting through FinnGen, 100 000 Genomes Project, and the National Health Service Genomic Medicine Service combined with clinical cohort from the Oulu University Hospital. Using IRD criteria from the International Classification of Diseases, 9th and 10th Revisions, 540 individuals with IRD and 473 945 control individuals were identified in the FinnGen study. For validation of FinnGen results, 49 patients were recruited from Oulu University Hospital. Results were further validated in 2 individuals identified from the UK cohort. MAIN OUTCOMES AND MEASURES: The GWAS and proteomics analysis were performed in the FinnGen cohort. Sanger and whole-genome sequencing and RNA approaches were used in a clinical IRD cohort to validate pathogenicity of the identified XXYLT1 variant. RESULTS: This GWAS identified 13 recessive loci reaching genome-wide significance (defined as P < 5 × 10-8). Of these, 4 (near or within XXYLT1, ANKRD10, DYM, and CBLN4) had not been associated with IRD, including the XXYLT1 c.505-1G>C founder variant. This variant was further genotyped in the clinical replication cohort, leading to identification of 5 more homozygous individuals from 4 families. The phenotype was consistent with a cone-rod or macular dystrophy, with visual deterioration, cystoid macular edema and/or schisislike macular abnormalities. The effect of the XXYLT1 c.505-1G>C variant was further investigated using RNA sequencing and complementary DNA amplicon sequencing, demonstrating exon 2 skipping and a loss-of-function effect. These findings were replicated in an independent population identifying 2 patients from the UK harboring a homozygous XXYLT1 c.766G>A, p.(Glu256Lys) missense variant. CONCLUSIONS AND RELEVANCE: This GWAS identified an association between XXYLT1 and IRD. These results affirm that GWAS in a founder population can be used as a potential tool for the discovery of rare mendelian disease genes and that XXYLT1 should be considered in clinical IRD gene panels.

Humans↗

PubMed2026

Doublesex gene influences sex differentiation and embryonic development in predatory mite Phytoseiulus persimilis.

BACKGROUND: Phytoseiulus persimilis is an effective biocontrol agent characterized by paternal genome elimination (PGE), an unusual reproductive system in which males eliminate the paternal genome during embryogenesis. However, the molecular mechanism underlying sex determination and reproductive regulation in this species remain poorly understood. RESULTS: Transcriptome-based analyses identified two doublesex (dsx) homologs, Ppdsx1 and Ppdsx2, as candidate regulators of reproduction. Weighted gene co-expression network analysis (WGCNA) assigned Ppdsx2 to a pre-mating-associated co-expression module enriched for reproductive and signaling pathways. Functional analyses revealed clear divergence between the two genes. RNA interference (RNAi) of Ppdsx1 reduced the proportion of female offspring, whereas RNAi of Ppdsx2 induced sex reversal, developmental abnormalities, and impaired egg viability. Yeast two-hybrid and glutathione S-transferase (GST) pull-down assays further demonstrated interactions between Dsx proteins and vitellogenin (Vg)-derived fragments identified from a complementary DNA (cDNA) library screen, suggesting a previously unrecognized connection between sex determination and reproductive nutrient allocation. CONCLUSIONS: Ppdsx1 contributes to maintenance of the female developmental pathway, whereas Ppdsx2 represents a strong candidate component of the PGE-associated sex-determination cascade. The observed Dsx-Vg fragment interaction suggests a potential link between reproductive developmental programs and nutrient allocation pathways. These findings provide new insights into the molecular basis of sex determination and reproductive regulation in phytoseiid mites and establish a foundation for future studies on the coupling of reproductive development and resource allocation. © 2026 Society of Chemical Industry.

Animals↗

PubMed2025

Expression patterns of plasma microRNAs in patients with cervical cancer from two teaching hospitals in Ghana.

AIM: Early cervical cancer diagnosis is a global challenge that needs to be addressed by the discovery of less invasive diagnostic and prognostic approaches. Circulating miRNAs are stable in plasma and their diagnostic potentials have been elucidated in some cancers. Therefore, in this cross-sectional study, we determined the patterns of expression of 7 selected circulating microRNAs that differ between patients with cervical cancer receiving therapy, patients with cervical not on therapy and healthy females. The goal was to investigate the diagnostic and prognostic potential of these selected miRNAs. METHODS: Total RNA was extracted from plasma samples collected from 53 participants recruited from Komfo Anokye Teaching Hospital and the Cape Coast Teaching Hospital, Ghana. Complementary DNA (cDNA) synthesis was performed, followed by quantitative polymerase chain reaction (qPCR) to amplify and quantify the expression levels of the target microRNAs. Expression levels of seven microRNAs-hsa-miR-146a, hsa-miR-29a, hsa-miR-29b, hsa-miR-34a, hsa-miR-233, hsa-miR-155, and hsa-miR-27a were compared among three groups: healthy controls (n = 27), patients with cervical cancer on therapy (n = 13), and those not on therapy (n = 13). RESULTS: miR-155 and miR-27a showed statistically significant differential expression between cancer patients and healthy controls. In addition, miR-29b expression levels differed significantly between stage 4b and stage 4a of patient with cervical cancer undergoing treatment. CONCLUSION: These findings suggest that circulating plasma miRNAs may serve as non-invasive biomarkers for the early detection of cervical cancer, monitoring disease progression, and evaluating treatment response.

Humans↗

PubMed2024

Nanopore sequencing to detect A-to-I editing sites.

Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA). However, such an approach is indirect and can be confounded by genomic single nucleotide polymorphisms (SNPs) and DNA mutations. Moreover, past studies rely primarily on the Illumina platform, which generates short sequencing reads that can be challenging to map. Recently, nanopore direct RNA sequencing has emerged as a powerful technology to address the issues. Here, we describe the use of the technology together with deep learning models that we have developed, named Dinopore (Detection of inosine with nanopore sequencing), to interrogate the A-to-I editome of any organism.

Inosine↗

PubMed2026

Systematic mapping of insertion-tolerant regions enables capsid engineering of an infectious RNA phage.

RNA phages are attractive platforms for the design of programmable bioparticles, but their development has been constrained by limited knowledge of genomic sites that can tolerate sequence insertion. Here, we combined MuA transposase-mediated in vitro insertion mutagenesis with our established reverse genetics systems to systematically identify insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. Screening of 4,555 MS2 and 2,228 PP7 random insertion clones identified 29 and 26 non-redundant ITRs, respectively. We further analyzed and compared these ITRs in the context of RNA genome organization and virion architecture. Both phages contained ITRs within the maturation protein, whereas only PP7 tolerated insertions within the coat protein (CP). On the basis of structural location and plaque-forming capacity, an ITR situated between Gly74 and Glu75 (GGC^GAG) in the PP7 CP was selected for further study. Infectious phage particles generated from complementary DNA clones retained the 15-bp insertion at both the RNA and protein levels. Engineered PP7 phages carrying an Arg-Gly-Asp motif inserted into the CP at this ITR displayed enhanced in vivo clearance in a Drosophila model, despite having in vitro stability comparable to that of the wild type. These findings provide the first example of CP engineering in an infectious RNA phage and establish a framework for engineering RNA phages for biological and biotechnological applications.IMPORTANCEA major obstacle to developing RNA phages as synthetic biology platforms is the lack of design principles for genomic insertion. Here, we address this limitation by establishing a mutagenesis-and-recovery workflow that systematically identifies insertion-tolerant regions (ITRs) in the RNA phages MS2 and PP7. The resulting maps reveal distinct structural constraints in the two phages and enable rational engineering of a peptide-display site in the PP7 capsid. Using this approach, we generated an engineered infectious phage with a modified capsid, thereby providing the first demonstration of capsid engineering in an infectious RNA phage, to our knowledge. This study lays the groundwork for the rational design of live RNA phage virions as tractable and engineerable scaffolds for future biological and biotechnological applications.

Animals↗

PubMed2005

SUMO modification of the Ets-related transcription factor ERM inhibits its transcriptional activity.

A variety of transcription factors are post-translationally modified by SUMO, a 97-residue ubiquitin-like protein bound covalently to the targeted lysine. Here we describe SUMO modification of the Ets family member ERM at positions 89, 263, 293, and 350. To investigate how SUMO modification affects the function of ERM, Ets-responsive intercellular adhesion molecule 1 (ICAM-1) and E74 reporter plasmids were employed to demonstrate that SUMO modification causes inhibition of ERM-dependent transcription without affecting the subcellular localization, stability, or DNA-binding capacity of the protein. When the adenoviral protein Gam1 or the SUMO protease SENP1 was used to inhibit the SUMO modification pathway, ERM-dependent transcription was de-repressed. These results demonstrate that ERM is subject to SUMO modification and that this post-translational modification causes inhibition of transcription-enhancing activity.

Adenoviridae↗

PubMed2026

DNA-aware evaluation and debiasing of sequence-to-function models.

MOTIVATION: Genome sequence-to-function (S2F) models are widely used to interpret base-resolution functional genomics assays. Most S2F models are trained and evaluated against observed counts and profile-shapes using statistical objectives and fidelity metrics. These choices are well motivated, but they are DNA-independent. At the same time, experimental measurements arise from DNA-dependent assays with distinct characteristics. This mismatch motivates a complementary DNA-aware evaluation of S2F-predicted and experimental functional genomic tracks. RESULTS: We study DNA-dependency of experimental and S2F-predicted tracks using track-conditional genome language models (cgLMs). cgLMs predict masked nucleotides from a conditioning track under controlled DNA visibility. Across ATAC-seq and TF ChIP-seq peaks from GM12878 and K562, cgLM-probing reveals a consistent masked DNA-decodability gap between many experimental and S2F-predicted tracks. In particular, single-task (e.g. BPNet) and multi-task (e.g. AlphaGenome) S2F-predicted tracks enabled cgLMs to recover masked nucleotides with significantly higher accuracy and confidence than matched experimental tracks. Analyses of nonpeak and dinucleotide-shuffled sequences show that this gap is not confined to peaks and is not captured by standard DNA-agnostic profile-shape fidelity metrics alone. ChromBPNet Tn5-denoised predictions were an exception and behaved closer to the experimental regime, suggesting that staged training may reduce the gap. We then convert this diagnostic into a critic-derived objective, DNA-dependency matching (DDM), using a frozen multi-headed cgLM critic. We introduce Critic-Guided Profile-Shape Editing (CGPSE), a preliminary post hoc debiasing framework for frozen S2F models. In GM12878 ATAC-seq, CGPSE partially reduces the masked DNA-decodability gap for AlphaGenome and BPNet predictions, while exposing a tradeoff with profile-shape fidelity. AVAILABILITY AND IMPLEMENTATION: https://github.com/li-lab-mcgill/dna-aware-s2f-eval.

DNA↗

PubMed2026

When R-Loops Go Awry: Genome Instability and Neurological Diseases.

The basic structure of DNA is a double helix formed by base pairing between complementary strands. However, during transcription, RNA hybridizes with the template DNA, whereas the complementary DNA strand becomes displaced and remains unpaired. This process forms a DNA-RNA hybrid structure known as an R-loop; similar structures can also occur in a non-co-transcriptional manner. In recent years, R-loops have been reported to be involved in various cellular functions. However, when not properly regulated, they can compromise genomic DNA stability. R-loops play roles in gene expression, DNA replication, and transcription termination. Dysregulation of R-loop homeostasis has been implicated in various human diseases, including neurological diseases. In this review, we discuss the physiological and pathological roles of R-loops, their related regulatory mechanisms controlling their formation and resolution, and their association with neurological diseases.

Humans↗

PubMed2005

Gene finding in the chicken genome.

BACKGROUND: Despite the continuous production of genome sequence for a number of organisms, reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularly true for genomes for which there is not a large collection of known gene sequences, such as the recently published chicken genome. We used the chicken sequence to test comparative and homology-based gene-finding methods followed by experimental validation as an effective genome annotation method. RESULTS: We performed experimental evaluation by RT-PCR of three different computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram was computed and each component of it was evaluated. The results showed that de novo comparative methods can identify up to about 700 chicken genes with no previous evidence of expression, and can correctly extend about 40% of homology-based predictions at the 5' end. CONCLUSIONS: De novo comparative gene prediction followed by experimental verification is effective at enhancing the annotation of the newly sequenced genomes provided by standard homology-based methods.

Animals↗

PubMed2025

Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High-Throughput Antiviral Screening Using Gibson DNA Assembly.

Coronaviruses (CoVs) pose a significant threat to human health, as demonstrated by the COVID-19 pandemic. The large size of the CoV genome (around 30 kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV-229E) of the genus Alphacoronavirus, mouse hepatitis virus A59 (MHV-59) of Betacoronavirus, and porcine deltacoronavirus (PDCoV-Haiti) of Deltacoronavirus. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome coronavirus (MERS-CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV-A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high-throughput drug screening. Thus, this study provides a practical and cost-effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.

Reverse Genetics↗

PubMed2025

Infectious Subgenomic Amplicon Strategies for Japanese Encephalitis and West Nile Viruses.

Classical methods for constructing infectious cDNA clones of flaviviruses are often hindered by instability and toxicity. The Infectious-Subgenomic-Amplicons (ISA) method is an advancement which utilizes overlapping DNA fragments representing viral genomic sequence and in-cell recombination to bypass bacterial plasmid assembly. However, the ISA method has limitations due to the toxicity of some ISA DNA fragments in bacteria during synthetic production. We validated modified ISA strategies for producing toxic ISA Japanese encephalitis virus (JEV) and West Nile virus (WNV) DNA fragments. Three approaches were explored, including subdividing toxic DNA fragments into two sub-fragments for synthetic clonal production, using a low-copy bacterial plasmid, and subdividing the toxic DNA fragments into four short overlapping sub-fragments, each up to 1.8 kb. The latter novel approach in ISA applications enabled the synthesis of entirely bacteria-free ISA DNA fragments. Our results demonstrate that subdividing toxic fragments into sub-fragments smaller than 1.8 kb for synthesis is the efficient strategy, circumventing the need for bacterial plasmids and ensuring rapid production of synthetic flaviviruses. This method also shortens the production timeline. We also compared the efficacy of JEV and WNV ISA in zinc finger antiviral protein 1 (ZAP) wild-type and knockout cells and found that knockout cells may be more effective for ISA rescue of flaviviruses, including CpG-enriched strains for live attenuated vaccines. The validated modified ISA strategies provide an efficient approach for producing synthetic JEV and WNV. This will enable rapid research during outbreaks of emerging flaviviruses by facilitating the quick generation of new virus variants.

West Nile virus↗

PubMed2027

Production of Viral Particles from a Chikungunya Virus Infectious Clone.

Chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus, which poses challenges for its study and genetic manipulation. Because direct mutagenesis of viral RNA genomes is technically impractical, reverse genetics systems are essential tools for investigating viral biology. To enable such approaches, infectious clones containing a full-length cDNA copy of the viral genome are constructed. The cDNA is positioned under the control of a bacteriophage RNA polymerase promoter, allowing commercial RNA polymerases to use the linearized plasmid as a template for the in vitro transcription of full-length viral genomic RNA (gRNA). Importantly, positive-sense viral genomes serve as mRNAs for the translation of viral proteins in a cellular environment, meaning that these transcripts contain all the information required to initiate viral replication. Following transfection into permissive cultured cells, viral proteins are expressed, enabling genome replication and, ultimately, the recovery of infectious particles from the cell supernatant. Here, we describe a detailed procedure for generating CHIKV particles through plasmid linearization, in vitro transcription, and subsequent RNA transfection.

Chikungunya virus↗

PubMed2025

Enzymes in high-throughput RNA sequencing: Applications and challenges.

High-throughput RNA sequencing provides genome-wide information on the dynamics of RNA in each cell and how the dynamics responds to environmental changes. Next-generation sequencing by the Illumina platform currently provides the highest information output as compared to other platforms. A key component of next generation sequencing of each RNA is the successful end-to-end reverse-transcription into a cDNA strand. This can be highly challenging given the propensity of each RNA to adopt ordered structures and to contain post-transcriptional modifications. While many reverse transcriptase (RT) enzymes have been developed over the years to maximize read-through of an RNA, their processivity and efficiency varies, raising the question of how to select the RT for the experiment at hand. Here, we use tRNA as a model for genome-wide sequencing, as tRNA has a stable secondary and tertiary structure and has a high density and wide variety of post-transcriptional modifications, presenting one of the most challenging problems of sequencing RNA. We compare the efficiency of end-to-end cDNA synthesis of tRNA among several recent RT enzymes and provide a general sequencing workflow that is applicable to most of these enzymes.

High-Throughput Nucleotide Sequencing↗