Journal: Nature Medicine

Article Title: Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

doi: 10.1038/s41591-022-01751-0

Figure Lengend Snippet: a , Representative liver histopathology sections stained with H&E (participants 1, 3, 4 and 11) or hematoxylin and Van Gieson (participant 15); histologic sections per participant were reviewed by the local pathologist and central pathologist, with consistent results: three biopsy levels (participant 1), two levels (participants 11 and 15), one level (participant 3) and four levels (participant 4). Images were captured in QuPath v0.2.3 using the export snapshot feature; no subsequent processing or image enhancement was performed. Scale bars, 180 µm (participant 1); 150 µm (participants 11, 15, 3 and 4) b , Representative liver biopsy sections from each participant showing hFVIII-SQ DNA (brown foci) by ISH. Images were captured at 1,600 × 1,200 pixels and output at 300 pixels per inch (ppi). Each focus (brown dot) represents at least one vector DNA molecule; it is possible to have multiple copies of vector genome within a single focus. Scale bars, 50 µm. c , Percentage of hepatocyte nuclei stained positive for hFVIII-SQ DNA by ISH. Data are means across 11 (participants 1 and 11) or 27–28 (participants 3, 4 and 15) images per biopsy section, spanning ≥50% of the tissue area (biopsy tissue area was larger for participants 3, 4 and 15). Error bars represent the s.e.m., dots represent quantification of each individual image, and data labels show mean values. d , Circular genomes (full length or H–T ITR fused) detected in liver biopsy samples via drop-phase ddPCR following DNA sample treatment with PS-DNase and KpnI (with PS-DNase, all linear forms of DNA are hydrolyzed, and only circular forms of DNA remain; KpnI treatment separates out vector genome units within the concatemeric forms, enabling quantification of genome units within concatemeric vector genomes). e , Qualitative Southern blot analysis of circular episomes after PS-DNase treatment of DNA from liver biopsy samples. Biopsy samples from control, participant 1 and participant 11, and for participants 3, 4 and 15 were processed at separate times; results are presented on separate blots from two independent experiments, and are not intended to present a quantitative comparison. kbp, kilobase pairs; SC, supercoiled markers; W, week.

Article Snippet: Before the droplet generation step of ddPCR, 5 µl of DNA at a concentration of 2 ng µl −1 was incubated with 4 units of high-fidelity KpnI (KpnI-HF) enzyme (New England Biolabs) in the ddPCR reaction mixture at 37 °C for 30 min. An additional set of samples was digested with PS-DNase (Lucigen) to hydrolyze all linear forms of DNA and isolate circular DNA.

Techniques: Histopathology, Staining, Plasmid Preparation, Southern Blot

Journal: Nature Medicine

Article Title: Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

doi: 10.1038/s41591-022-01751-0

Figure Lengend Snippet: Quantification of circular vector genomes [DNA] as measured by ddPCR utilizing primers and probes to detect specific regions of the vector genome: a) R2-R10 linked amplicons. b) R1-R11 linked amplicons (full-length). c) SQ amplicon (representing overall vector genome count). d) ITR-fused amplicons; ITR fusion assay measured 5′ to 3′ ITR recombination (that is head-to-tail, H-T). e) Circular multimeric episome quantification following PS-DNase (eliminates all linear forms of DNA) or PS-DNase/KpnI digestion (to separate individual vector genome units within concatemeric circular forms). ITR fusion assay measured 5′ to 3′ ITR recombination (that is head-to-tail, H-T). f) Qualitative Southern blot assessment of vector genome configuration in circular forms obtained after DNA treatment with PS-DNase and KpnI. DNA fragments corresponding to 3.5 kb, or 4.4 and 2.6 kb could be detected for H-T or H-H/T-T concatemer configurations, respectively. Biopsies from Control, Participant 1, and Participant 11, and for Participants 3, 4, and 15 were processed at separate times; results are presented on separate blots from two independent experiments, and are not intended to present a quantitative comparison. ddPCR, droplet digital polymerase chain reaction; H, head (5′ end); H-H, head-to-head orientation; H-T, head-to-tail orientation; ITR, inverted terminal repeat; kb, kilobase pairs; LM, linear markers; PS-DNase, Plasmid Safe™ ATP-Dependent DNAase; T, tail (3′ end); vg, vector genome. ddPCR was performed after DNA digestion with PS-DNase and KpnI (to eliminate linear forms and to separate individual vector genome units within concatemeric circular forms).

Article Snippet: Before the droplet generation step of ddPCR, 5 µl of DNA at a concentration of 2 ng µl −1 was incubated with 4 units of high-fidelity KpnI (KpnI-HF) enzyme (New England Biolabs) in the ddPCR reaction mixture at 37 °C for 30 min. An additional set of samples was digested with PS-DNase (Lucigen) to hydrolyze all linear forms of DNA and isolate circular DNA.

Techniques: Plasmid Preparation, Amplification, Single Vesicle Fusion Assay, Southern Blot, Digital PCR

Journal: Cell host & microbe

Article Title: Antiretroviral therapy reveals triphasic decay of intact SIV genomes and persistence of ancestral variants.

doi: 10.1016/j.chom.2023.01.016

Figure Lengend Snippet: Figure 4. Comparison of gag ddPCR and IPDA quantification of SIV DNA (A) Correlation between gag+ SIV DNA copies and intact proviruses quantified using the IPDA. Individual datapoints represent the number of copies detected using either an amplicon in gag (y-axis)65 or the IPDA (x-axis).20 Each point is the geometric mean of 3 replicates. Intact proviruses are corrected for shearing (DSI) and env+2LTR circles. The correlation between the two variables was calculated using Pearson’s coefficient. Data from one animal, T624, were excluded from this analysis due to failure of the gag amplicon resulting from mutations or deletion. (B) Correlation between gag+ SIV DNA copies and intact proviruses, with gag values corrected using the same env+2LTR correction factor applied to the IPDA data, calculated using Pearson’s coefficient. (C) Comparison of the decay of SIV gag copies and intact proviruses for the animals in cohort 18–02. IPDA data are corrected for env+2LTR circles and DNA shearing. Vertical lines represent the standard deviations. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains SIVmac251 swarm 56 Dr. Dan Barouch, BIDMC N/A Biological samples N/A SIVmac251-infected Rhesus macaque PBMCs Dr. Dan Barouch, BIDMC N/A Chemicals, peptides, and recombinant proteins Platinum TaqDNA Polymerase High Fidelity ThermoFisher Scientific Cat # 11304011 SuperScript III Reverse Transcriptase ThermoFisher Scientific Cat # 18-080-044 dNTP Mix (10 mM each) ThermoFisher Scientific Cat # 18-427-088 UltraPure 1M Tris-HCl Buffer (pH 8.0) ThermoFisher Scientific Cat # 15-568-025 RNaseOUT Recombinant Ribonuclease Inhibitor ThermoFisher Scientific Cat # 10777019 Critical commercial assays QIAamp DNA Mini Kit Qiagen Cat # 51304 CD4+ T Cell Isolation Kit, Non-Human Primate Miltenyi Biotec Cat # 130-092-144 2X ddPCR Supermix for Probes (no dUTPs) Bio-Rad Cat # 186-3024 Deposited data SIV – plasma RNA sequences This paper Genbank: OQ168641-OQ168979 SIV – non-defective proviral DNA sequences This paper Genbank: OQ168980-OQ170751 SIV – hypermutated (defective) proviral sequences This paper Genbank: OQ170752-OQ170785 Experimental models: Organisms/strains Rhesus macaque (Macaca mulatta) infected with SIVmac251 Indian origin Animals T523, T530, T537, T544, T545, T623, T624, T625, T627 & T628 Oligonucleotides Oligonucleotides, standard desalting (Sanger sequencing & ddPCR) – see STAR Methods and Table S8 IDT N/A FAM/VIC labeled probes with MGB quencher, HPLC purified (see Table S8) Applied Biosystems N/A Unlabeled competition probes w/MGB quencher, HPLC purified (see Table S8) Applied Biosystems N/A FAM/HEX labeled probes w/MGB-NFQ quencher, HPLC purified (see Table S8) IDT N/A Recombinant DNA Synthetic double-stranded DNA controls (gBlocks) – see STAR Methods IDT N/A Software and algorithms QuantaSoft Analysis-Pro Bio-Rad http://www.bio-rad.com/en-us/ product/qx200-droplet-digitalpcr-system?ID=MPOQQE4VY Geneious Prime Dotmatics https://www.geneious.com/prime/ MEGA v7.0 & v11 MEGA https://www.megasoftware.net/ (Continued on next page) Cell Host & Microbe 31, 356–372.e1–e5, March 8, 2023 e1

Techniques: Comparison, Amplification

Journal: Immunity

Article Title: Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function

doi: 10.1016/j.immuni.2020.07.006

Figure Lengend Snippet:

Article Snippet: Amplification and quantification were performed on a QX100 Droplet Digital PCR system (BioRad).

Techniques: Recombinant, Molecular Cloning, Blocking Assay, Conjugation Assay, Staining, Western Blot, Lysis, Mutagenesis, Isolation, Luminex, Transfection, Amplification, Software, Variant Assay

Journal: bioRxiv

Article Title: High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19

doi: 10.1101/2021.02.21.432184

Figure Lengend Snippet: (A) SARS-CoV-2 genomic RNA (gRNA) is reverse-transcribed to include an 8-nucleotide unique molecular identifier (UMI; multicolored bar), followed by PCR amplification and Pacific Biosciences single-molecule, real-time (SMRT) sequencing of the 6.1-kilobase region encompassing spike (S), ORF3, envelope (E), and membrane (M) protein genes. After quality control and trimming, sequence reads are compiled into bins that share a UMI sequence, and bins with low read counts are removed according to the inflection point of the read count distribution (see ). Presumptive false bins arising from errors in the UMI are then identified and removed by the network adjacency method, followed by further removal of bins with the lowest read counts using a more conservative knee point cutoff (see ). Variant calling is then used to identify presumptive erroneous mutations based on rarity and pattern (ex., single-base insertions adjacent to homopolymers), and these are reverted to the sample consensus. Finally, SGS that correspond to haplotypes occurring only once in each sample are excluded (not pictured). (B) To validate data generation and analysis procedures, clonal RNAs transcribed in vitro from USA/WA-1 and double mutant sequences were mixed at varying ratios and subjected to HT-SGS. Results are described in Results and .

Article Snippet: The QX200 AutoDG Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA) was used to detect and quantify SARS-CoV-2 RNA using the SARS-CoV-2 Droplet Digital PCR Kit (Bio-Rad), which contains a triplex assay of primers/probes aligned to the CDC markers for SARS-CoV-2 N1 and N2 genes and human RPP30 gene.

Techniques: Amplification, Sequencing, Variant Assay, In Vitro, Mutagenesis

Journal: bioRxiv

Article Title: High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19

doi: 10.1101/2021.02.21.432184

Figure Lengend Snippet: (A) Haplotype diagrams (left) depicting SARS-CoV-2 SGS detected in a 4 th -passage Vero cell culture of the WA-1 reference clinical isolate. Spike NH 2 -terminal domain (NTD), receptor-binding domain (RBD), and furin cleavage site (F) regions are shaded grey, with remaining regions of spike in white. Pink tick marks illustrate mutations relative to the sample consensus sequence. Amino acid changes corresponding to these mutations are shown in sequence alignment form (middle), with the percentage of all SGS in the sample matching each haplotype shown in the bar graph (right). The grey bar in the graph indicates the haplotype that matches the sample consensus sequence; variant haplotypes with at least 1 mismatch to sample consensus are in pink. (B) Read counts of each UMI bin for which the SARS-CoV-2 sequence matched each of 18 different haplotypes in Vero cell culture of the WA-1 clinical isolate. Bars indicate median read counts among bins. (C) Mapping of detected spike gene mutations on the trimer structure. Two protomers of the SARS-CoV-2 spike (PDB ID: 6zge) are shown in surface representation and colored light blue and wheat, respectively. The third protomer is shown in cartoon representation with the NTD region colored in bright green. NTD mutations as well as T307I and H655Y are shown in red and the furin cleavage site mutations are in brown. The molecular structures were prepared with PyMOL ( https://pymol.org ).

Article Snippet: The QX200 AutoDG Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA) was used to detect and quantify SARS-CoV-2 RNA using the SARS-CoV-2 Droplet Digital PCR Kit (Bio-Rad), which contains a triplex assay of primers/probes aligned to the CDC markers for SARS-CoV-2 N1 and N2 genes and human RPP30 gene.

Techniques: Cell Culture, Binding Assay, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19

doi: 10.1101/2021.02.21.432184

Figure Lengend Snippet: Each participant label indicates day of clinical illness and the number of SGS obtained for the sample in parentheses. Haplotype diagrams (left) depicting SARS-CoV-2 SGS are as in . Non-synonymous or synonymous mutations in each haplotype relative to the WA-1 reference sequence are shown with pink or blue tick marks. Amino acid changes (middle) and percentages of all SGS in the sample attributable to indicated haplotypes (right) are as in . The haplotype matching the consensus for each sample is represented in grey; variant haplotypes with at least 1 non-synonymous mismatch to sample consensus are in pink.

Article Snippet: The QX200 AutoDG Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA) was used to detect and quantify SARS-CoV-2 RNA using the SARS-CoV-2 Droplet Digital PCR Kit (Bio-Rad), which contains a triplex assay of primers/probes aligned to the CDC markers for SARS-CoV-2 N1 and N2 genes and human RPP30 gene.

Techniques: Sequencing, Variant Assay

Journal: bioRxiv

Article Title: High-Throughput, Single-Copy Sequencing Reveals SARS-CoV-2 Spike Variants Coincident with Mounting Humoral Immunity during Acute COVID-19

doi: 10.1101/2021.02.21.432184

Figure Lengend Snippet: (A) Copy numbers of SARS-CoV-2 N1 (black squares) and N2 (grey circles) RNA (left y-axis) and percentage of SGS not matching the predominant/consensus haplotype (pink diamonds, right y-axis) plotted for upper respiratory tract samples from days 9–17. (B) Variant haplotypes of the SARS-CoV-2 virion surface protein gene region detected on days 9, 11, 13, 15, and 17. The number of SGS obtained at each day is in parentheses. Haplotype diagrams (left), amino acid changes (middle), and percentages of all SGS in the sample attributable to indicated haplotypes (right) are as in and . The haplotype matching the consensus for each sample is represented in grey; variant haplotypes with at least 1 non-synonymous mismatch to sample consensus are in pink; one variant haplotype differing from sample consensus by only a synonymous mismatch is in blue. (C) Mapping of detected spike gene mutations on the trimer structure, viewed from the side (left) and top (right). The protomers in the spike (PDB ID: 6zge) were shown and colored with the same scheme as in . Detected mutations are highlighted in red. Antibody 4A8 (PDB ID: 7c21) is shown to bind to NTD with its epitope (blue) overlapping with the detected NTD mutations (right). The molecular structures were prepared with PyMOL ( https://pymol.org ). (D) Relative contribution of NTD epitope-specific serum antibodies to total NTD domain-specific binding on days 9, 12, 16, and 19. Plotted results represent averages of 2–4 replicate experiments for each condition.

Article Snippet: The QX200 AutoDG Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA) was used to detect and quantify SARS-CoV-2 RNA using the SARS-CoV-2 Droplet Digital PCR Kit (Bio-Rad), which contains a triplex assay of primers/probes aligned to the CDC markers for SARS-CoV-2 N1 and N2 genes and human RPP30 gene.

Techniques: Variant Assay, Binding Assay