Panoramic Parvovirus Strain Discovery Data Chain

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ParvoDB ID PV027089 GenBank Accession MZ708675
Subfamily Parvovirinae Genus Dependoparvovirus
Species unclassified Dependoparvovirus Sequence Length 2211
Definition Adeno-associated virus isolate KN59_S59 capsid protein VP1 (VP1) gene, complete cds. Submission Date 2021-08-04
Strain Name KN59_S59 Sampling Date 2018
Sampling Country United States of America Location United States of America
Submitted By Wang,Q., Nambiar,K. and Wilson,J.M. Submitting Institution Gene Therapy Program, University of Pennsylvania, 125 S. 31st Street, Suite 1200, Philadelphia, PA 19104, USA
Original Host Homo sapiens Sample Type tissue samples
Standardized Host Name Homo sapiens Host Tax Rank species
Standardized Host Class Mammalia Standardized Host Order Primates
Environmental Origin - N/A - Reagent Origin - N/A -
Number Reagent/Consumables Purpose PMID/Url Reference
1 QIAamp DNA Mini Kit (QIAGEN Inc., Germantown, MD) Genomic DNA extraction from nonhuman primate and human tissue samples. 34448594 View Related Publication
2 Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA) PCR amplification of 3.1 kb AAV genome sequences from host genomic DNA with high accuracy.
3 'AV1NS' forward primer and 'AV2CAS' reverse primer Isolate AAV genomes during PCR; replaced the degenerate base Y in AV1NS with T.
4 AV1NS’ 5’-GCTGCGTCAACTGGACCAATGAGAAC-3’ Forward primer for PCR-based AAV genome isolation.
5 AV2CAS 5’-CGCAGAGACCAAAGTTCAACTGAAACGA-3’ Reverse primer for PCR-based AAV genome isolation.
6 TOPO cloned (Thermo Fisher Scientific, Waltham, MA) Clone PCR products for Sanger sequencing.
7 Salmon sperm DNA (Ambion, Inc., Austin, TX) Used for endpoint dilution in AAV-Single Genome Amplification (SGA) from human tissue samples.
8 Agencourt Ampure XP Beads (Beckman Coulter, Brea, CA) Purification of AAV DNA amplicons from PCR reactions.
9 NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, Ipswich, MA) Construction of libraries for sequencing using the Illumina MiSeq 2x250 paired-end sequencing platform.
10 Illumina MiSeq 2x250 (Illumina, San Diego, CA) Sequencing platform used for paired-end sequencing of AAV DNA amplicons.
11 HotStar HiFidelity polymerase (QIAGEN Inc.) Conduct comparative studies with Q5 polymerase; known for lower fidelity in PCR amplifications.
12 Stable Competent Escherichia coli cells (Thermo Fisher Scientific) Transformation for creating pure template stocks for subsequent experiments.
13 McapF3SpeI (5’-ATCGATACTAGTCCATCGACGTCAGACGCGGAA-3’) Primer used in the second round of nested PCR for AAV genome amplification.
14 McapR1NotI’ (5’-ATCGATGCGGCCGCAGTTCAACTGAAACGAATTAAACGGT-3’) Primer used in the second round of nested PCR for AAV genome amplification.
15 PvuII (New England Biolabs) Restriction enzyme used for linearizing plasmids as PCR templates.
16 HotStar polymerase Used in comparative fidelity studies; found to introduce significant mutations in AAV sequences.
17 HEK293 cells Used in vector production and transfection assays.
18 Triple-transfection cell lysates Vector harvesting for DNA-vector titration studies.
19 Firefly luciferase gene (CB7.ffluciferase.rBG) Transgene packaged in AAV vector to measure infectivity and vector production efficiency.
20 Geneious Prime version 2019.2 Used for constructing DNA sequence alignments and phylogenetic analyses (Note: This is software mentioned here just for context but excluded from the final list).