Journal: Molecular Therapy. Nucleic Acids

Article Title: Orthogonal characterization of rAAV reveals vector attributes that drive ITR repair, self-complementary genome formation, and transgene expression

doi: 10.1016/j.omtn.2026.102899

Figure Lengend Snippet: Recombinant AAV genome element and heterogeneity analysis by native and alkaline electrophoresis (A) Graphic representation of the main recombinant AAV vector genome elements evaluated: wtITR (143 nt, black) versus dITR/wtITR (11-nt deletion in the 5′ C arm, 130 nt, red), EAlbAAT (albumin enhancer-alpha1-antitrypsin promoter, blue), Egfp (enhanced green fluorescent protein, green), pA (bovine/human growth hormone polyadenylation signal, yellow), and CBA (CMV (cytomegalovirus) enhancer-chicken beta actin promoter, orange). Vector genome sizes are shown next to each representation. (B) Native automated electrophoresis of extracted rAAV genomes from dITR/wtITR and wtITR. EalbAAT . Egfp vectors by TapeStation. From left to right: AAV2, AAV3B, AAV5, AAV8, AAV9, and AAVLK03 serotypes. As a run reference, the upper marker (10,000 bp) and lower marker (15 bp) of the ladder are highlighted in purple and green, respectively. (C) Native automated electrophoresis of extracted rAAV genomes from AAV5 and AAV8 serotypes with dITR vs. wtITR and CBA versus EalbAAT promoters. As a run reference, the upper marker (10,000 bp) and lower marker (15 bp) of the ladder are highlighted in purple and green, respectively. (D) Native automated electrophoresis of extracted rAAV genomes from the AAVLK03 serotype with dITR and CBA vs. EalbAAT promoters. As a run reference, the upper marker (10,000 bp) and lower marker (15 bp) of the ladder are highlighted in purple and green, respectively. (E) Native electrophoresis of extracted rAAV genomes from AAV9 and AAVLK03 serotypes with dITR and CBA versus EalbAAT promoters, before and after digestion at 37°C for 1 h with SmaI. (F) Graphic representation of the dITR/tITR design (11-nt deletion in the 5′ C arm and 22-nt deletion of the 3′ B arm, 121 nt, purple), scITR (C arm deletion, 106 nt, green), and the dITR. AAT . Egfp expression cassette including an hypoxanthine phosphoribosyltransferase 1 ( HPRT 1 ) DNA stuffer sequence (GenBank: NG_012329.2 ) (bottom). Vector genome sizes are shown next to each design. (G) Alkaline electrophoresis of AAV8 vectors with different ITR configurations and CBA versus EalbAAT promoters. (H) Alkaline electrophoresis of AAV8 and AAV3B vectors with dITR/wtITR, dITR/tITR, and scITR configurations. (I) Alkaline electrophoresis of AAV8 and AAVLK03 vectors with the dITR/tITR configuration. (J) Alkaline electrophoresis of AAV3B, AAV5, and AAVLK03 vectors with dITR/wtITR configuration and CBA versus EalbAAT promoters. Combinations are displayed on top of each lane. Agarose gel ladder: FastGene 1 kb DNA marker (10,000–100 bp).

Article Snippet: For the SMN1 vectors, AAV genome integrity was analyzed using the AAV sequencing service provided by Plasmidsaurus Inc. (Eugene, OR, USA).

Techniques: Recombinant, Electrophoresis, Plasmid Preparation, Marker, Expressing, Sequencing, Agarose Gel Electrophoresis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Orthogonal characterization of rAAV reveals vector attributes that drive ITR repair, self-complementary genome formation, and transgene expression

doi: 10.1016/j.omtn.2026.102899

Figure Lengend Snippet: Recombinant AAV genome element effect and heterogeneity analysis by long-read single molecule, real-time SMRT sequencing (A) Read counts and length of rAAV3B and rAAVLK03 vector genomes encoding the Egfp transgene with dITR, dITR/tITR, and scITR designs, and AAT versus CBA promoters. (B) Identification of vector genome start and end sequencing positions from the AAV3B and AAVLK03 constructs (displayed on top). Start positions are labeled in blue, and end positions are labeled in red. Quantification of read counts is displayed in the Y axis. Vector genome elements are displayed on top as a reference. (C) Read counts and length of rAAV8 vector genomes encoding the Egfp reporter transgene with different ITR designs (from left to right: wtITR, dITR/wtITR, dITR/tITR, and scITR) under the control of the EAlbAAT or CBA promoters. The dITR.AAT. Egfp . Stuffer vector is also included. (D) Identification of vector genome start and end sequencing positions from the aforementioned AAV8 constructs. Start positions are labeled in blue, and end positions are labeled in red. Quantification of read counts is displayed on the Y axis. Vector genome elements are displayed on top as a reference.

Article Snippet: For the SMN1 vectors, AAV genome integrity was analyzed using the AAV sequencing service provided by Plasmidsaurus Inc. (Eugene, OR, USA).

Techniques: Recombinant, Sequencing, Plasmid Preparation, Construct, Labeling, Control

Journal: The Journal of Cell Biology

Article Title: Insights into retinal disease and non-tubulin glutamylation from a RPGR–TTLL5 complex structure

doi: 10.1083/jcb.202508020

Figure Lengend Snippet: A minimal C-terminal helix in the RPGR BD is sufficient for CID binding and intercalates into the CID helical bundle through a conserved aromatic network. (A) Domain organization of human TTLL5 and RPGR. TTL catalytic core, blue; cMTBD, maroon; CID, green. RCC1-like β-propeller domain in RPGR, yellow, with each repeat shown as a separate propeller blade. Sequence for the minimal BD peptide required for TTLL5 targeting to RPGR shown in magenta at the top. (B) ITC measurements of WT miniBD peptide titrated into CID. Best fit of n = 2 independent experimental replicates. (C) 2.8-Å X-ray crystal structure of human TTLL5 CID in complex with human RPGR miniBD shown in cartoon representation; CID, green; RPGR, magenta. (D and E) Key conserved hydrophobic interactions at the CID–BD interface. Solid black boxes around residue labels designate disease-associated mutations, dashed boxes designate mutations without disease annotation in the ClinVar database that we predict are pathogenic based on our structure. RPGR and TTLL5 are colored as in B; H-bonds are shown as dashed lines.

Article Snippet: Vectors were sequenced by Psomagen standard Sanger sequencing service.

Techniques: Binding Assay, Sequencing, Residue

Journal: The Journal of Cell Biology

Article Title: Insights into retinal disease and non-tubulin glutamylation from a RPGR–TTLL5 complex structure

doi: 10.1083/jcb.202508020

Figure Lengend Snippet: Multiple sequence alignment of vertebrate TTLL5 CID and RPGR BD. (A) TTLL5 CID. (B) RPGR miniBD. Secondary structure elements indicated above the sequence and based on our X-ray crystal structure of the complex; gaps of disordered, unmodeled residues are indicated by a dotted line. Hydrophobic residues at the CID–BD interface indicated with blue boxes. Residues mutated for ITC experiments indicated with black arrows above the sequence.

Article Snippet: Vectors were sequenced by Psomagen standard Sanger sequencing service.

Techniques: Sequencing

Journal: Biofilm

Article Title: Experimental evolution in the cystic fibrosis chemical environment reveals early TCA cycle flux as a central regulator of Mycobacterium abscessus biofilm formation

doi: 10.1016/j.bioflm.2025.100343

Figure Lengend Snippet: Experimental evolution of MAB 0253a in SCFM1 selects for novel biofilm phenotypes. A) Graphical overview of the evolution experiment. SNG stands for S CFM1 n o g lucose B) Colony morphologies of all evolved populations from passage 15 of the evolution experiment on TYEM agar plates. The smooth variant MAB 0253a (ancestor) and the rough variant MAB 0253b are included for comparison. C) Aggregation dynamics of all evolved populations from passage 15 of the evolution experiment in TYEM media. The left plot depicts the peak aggregate OD 600 measurement of each lineage, taken within 40–52 h post-inoculation. The right plot depicts the aggregate OD 600 readout taken at 68 h post-inoculation. P-value obtained by unpaired Student's T-test against the Ancestor. ∗ denotes P-value <0.05. D) Graphical overview of MAB_0812 ( mraA ) and MAB_0813c ( mraB ) on the positive and negative strands, with nucleotide positions on the contiguous genome sequence labelled on the horizontal axis. Mutations observed in laboratory evolution experiments are denoted with a blue point. The helix-turn-helix (HTH) DNA-binding domain is green and the C-terminal effector-binding domain is red. Allelic variants for orthologs isolated from people with cystic fibrosis are denoted with red points and are labelled with their amino acid positions on each gene. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: PCR products with the correct fragment size were purified and sequenced using the Sanger Sequencing Services at Azenta Life Sciences.

Techniques: Variant Assay, Comparison, Sequencing, Binding Assay, Isolation

Journal: PLOS One

Article Title: Sequencing complete plasmids on Oxford Nanopore Technologies sequencers using R2C2 and Chopper

doi: 10.1371/journal.pone.0345168

Figure Lengend Snippet: Plasmids were processed with rolling circle amplification (RCA) to produce linear DNA containing multiple tandem repeats of the original plasmids. The linear DNA was prepped and sequenced on an ONT P2Solo. The resulting ONT data were then processed into ONT reads by dorado and consensus reads by an updated version of C3POa. Consensus reads and subreads originating from multiple reads covering the same plasmid were then combined into one highly accurate plasmid sequence using the newly developed Chopper.

Article Snippet: To determine if commercial plasmid sequencing services would struggle with these homopolymers as well, we sent all four plasmids in this study to Plasmidsaurus for sequencing and analysis.

Techniques: Amplification, Plasmid Preparation, Sequencing

Journal: PLOS One

Article Title: Sequencing complete plasmids on Oxford Nanopore Technologies sequencers using R2C2 and Chopper

doi: 10.1371/journal.pone.0345168

Figure Lengend Snippet: For each sequencing run, the read length distributions of the raw ONT sequencing data (grey) and R2C2 consensus reads (blue) are shown as histograms. The length of sequenced plasmids (orange) included in each run is also shown.

Article Snippet: To determine if commercial plasmid sequencing services would struggle with these homopolymers as well, we sent all four plasmids in this study to Plasmidsaurus for sequencing and analysis.

Techniques: Sequencing

Journal: PLOS One

Article Title: Sequencing complete plasmids on Oxford Nanopore Technologies sequencers using R2C2 and Chopper

doi: 10.1371/journal.pone.0345168

Figure Lengend Snippet: Chopper accepts R2C2 consensus reads and their subreads produced by C3POa as input. It then bins R2C2 consensus reads by length to identify different plasmids in the sequencing data. For each length bin/plasmid, Chopper then generates a highly accurate plasmid sequence using racon and medaka for polishing.

Article Snippet: To determine if commercial plasmid sequencing services would struggle with these homopolymers as well, we sent all four plasmids in this study to Plasmidsaurus for sequencing and analysis.

Techniques: Produced, Sequencing, Plasmid Preparation

Journal: PLOS One

Article Title: Sequencing complete plasmids on Oxford Nanopore Technologies sequencers using R2C2 and Chopper

doi: 10.1371/journal.pone.0345168

Figure Lengend Snippet: Chopper was run on data from the 4 sequencing runs on individual plasmids. 100 R2C2 consensus reads were polished for each plasmid sequencing run (using the --iterations argument). Each R2C2 consensus read was polished with increasing numbers of R2C2 subreads (using the --subreads argument). With the exception of pSpCas9, Chopper produced almost entirely error-free sequences once more than 20 R2C2 subreads were used for polishing.

Article Snippet: To determine if commercial plasmid sequencing services would struggle with these homopolymers as well, we sent all four plasmids in this study to Plasmidsaurus for sequencing and analysis.

Techniques: Sequencing, Plasmid Preparation, Produced

Journal: PLOS One

Article Title: Sequencing complete plasmids on Oxford Nanopore Technologies sequencers using R2C2 and Chopper

doi: 10.1371/journal.pone.0345168

Figure Lengend Snippet: R2C2 subreads, R2C2 consensus reads, and Chopper sequences are aligned to the indicated plasmid references. Mismatches and Indels and are indicated in red, alignment ends in black B) . The error-rate of R2C2 consensus reads and Chopper sequences at specific positions of the pSpCas9 plasmid. These error-rates are shown as stacked bargraphs for a region of the plasmid that contains two homopolymers which represent a source for systematic sequencing error. C) Pairwise alignment of the pSpCas9 plasmid reference and the sequence Plasmidsaurus produced for that plasmid.

Article Snippet: To determine if commercial plasmid sequencing services would struggle with these homopolymers as well, we sent all four plasmids in this study to Plasmidsaurus for sequencing and analysis.

Techniques: Plasmid Preparation, Sequencing, Produced