AAV Capsid Barcode Library

AAV Capsid Barcode Library

At AAVnerGene, we empower researchers to accelerate AAV capsid discovery through our advanced AAV Capsid Barcode Library Services. Barcode-Seq utilizes deep-sequencing technologies such as Illumina amplicon-seq to track the performance of individual AAV vector within complex mixtures. In this approach, each AAV capsid variant encapsulates an identical transgene expression construct that differs only by a short, unique DNA barcode. This barcode serves as a molecular identifier, allowing precise quantification of each variant’s abundance and transduction efficiency across different tissues or cell types. To minimize potential barcode-dependent bias, each variant can be linked to multiple independent barcodes, ensuring statistically robust and reproducible results. Designed for capsid evolution and tissue tropism studies, these kits enable parallel evaluation of hundreds or thousands of AAV capsid variants in vivo or in vitro. Researchers can determine which variants show superior packaging efficiency, transduction, or tissue specificity — all in a single sequencing run.

Design of AAV Capsid Barcode Library

In the ATHENA-I, each capsid variant is linked to three unique DNA barcodes, all carrying the same reporter gene. This reduces variability and improves accuracy in high-throughput screening. For example, The AAV Capsid Barcode Kit-CAG-EGFP includes AAV genomes with a CAG promoter driving EGFP expression, followed by the WPRE enhancer and bGH PolyA signal. The DNA barcode is placed between the WPRE and bGH PolyA. The CAG promoter ensures strong expression across cell types, while the EGFP reporter gene helps identify and isolate successfully transduced cells. WPRE and bGH PolyA enhance EGFP expression and stabilize mRNA.

AAVnerGene offers end-to-end services for the custom construction and production of AAV Capsid Barcode Library. Customers can tailor their libraries by selecting their preferred promoter, reporter gene, barcode design, and capsid variants, ensuring fully customized solutions to meet specific experimental needs. Customization Options:

• Select Your Capsids: Choose from a diverse set of capsids representing various serotypes, tropisms, and transduction efficiencies. Options include well-characterized AAV serotypes, newly engineered capsids, or variants with specific properties.
• Choose Your Promoter: Select the optimal promoter to ensure your gene of interest is expressed in the right cells, at the desired level, and for the required duration.
• Select Your Reporter System: Pick a reporter gene or cassette for easy detection and quantification of transduction efficiency and tropism. Common options include:
  • Fluorescent proteins: GFP, mCherry, etc.
  • Enzymatic reporters: Luciferase (Cluc, Fluc, Gluc, Rluc), β-galactosidase, etc.
• Design Your DNA Barcode Strategy: Incorporate unique DNA barcodes to track and identify individual capsid variants. Barcodes are typically placed in non-coding regions and designed for compatibility with downstream sequencing or detection methods.
  • Barcode Design Options: N25, 2xN12
  • Number of Barcodes per Capsid: We recommend using 3 barcodes per capsid to balance cost and experimental accuracy.
• Determine Production Strategy and Scale: Plan the production strategy and scale based on the number of capsid variants, desired yields, and intended applications.
  • Individual Purification: 1e12 vg, 2e12 vg, 5e12 vg, 1e13 vg, etc.
  • Pooled Purification: 10 cm dish, 15 cm dish, 850 cm² roller bottle,  125ml flask, 1L flask etc.

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Production of AAV Capsid Barcode Library

Unlike custom AAV Capsid Barcode Kits, the AAV Capsid Barcode Library includes a larger number of AAV capsid variants, and the library is purified and provided as pooled particles.  The production process involves the following steps:

• Plasmid Preparation: The selected pRCap plasmid (encoding the AAV capsid) is paired with requested AAV barcode plasmids (each containing a unique DNA barcode).
• Co-Transfection: The paired plasmids are co-transfected into HEK 293T/HEK 293 or our HEK 293one cells using our PEIone transfection reagent in our standard protocol to produce AAV particles.
• AAV Purification and Titration: The produced AAVs are pooled and purified using advanced purification methods to ensure high-quality yields. The final AAV capsid barcode library is then titrated to determine its concentration and ensure batch-to-batch consistency.
• Pooling for Injection: The pool AAV vectors can be directly for in vitro or in vivo injections.

Price and Turnaround

The production cost is based on the production scales and requested barcode number per capsid. 

Production Scale	Price/Capsid				Bulk Order Discount	Turnaround  Time
Barcodes/Capsid	3	4	5	More	10~20, 10%; 21-40, 15%; 41~70, 20%; 71-100, 25%; >100, request	3-4 weeks
25 ml culture or 15-cm dish	$400	$450	$500	Quote
100 ml culture or 850-cm2 roller bottle	$1,000	$1,100	$1,200	Quote

1. The listed price covers packaging services only;

2.Additional costs will apply if plasmid preparation is required

3. We provide both mini-helper plasmid and barcode AAV transgene plasmids;

AAV Barcode Plasmids

AAV barcode reporter plasmids are essential for generating AAV Capsid Barcode Kits. Each plasmid contains a unique barcode strategically positioned between the WPRE enhancer and the PolyA sequence. At AAVnerGene, we offer a diverse range of AAV barcode plasmids featuring various backbones, promoters, reporters, and barcodes to meet your specific research needs. Researchers can order sets of 100 AAV barcode plasmids, each containing unique barcode, directly from AAVnerGene.

Backbone	Product Name	SKU	Barcode Design	Request Plasmids
pAAVtri	pAAV-CAG-EGFP-BC-N25	SA005001A	N25 (NNNNNNNNNNNNNNNNNNNNNNNNN)	pRCap+  mini-pHelper
	pAAV-CAG-EGFP-BC-2XN12	SA005001B	2XN12(NNNNNNNNNNNNCGGAAATACGATGTCGGGANNNNNNNNNNNN	pRCap + mini-pHelper
pAAVdual	pAAVdual-CMV-mCherry-BC-N25	SA001006A	N25 (NNNNNNNNNNNNNNNNNNNNNNNNN)	pRcap
	pAAVdual-sc-CMV-mCherry-BC-N25	SA023006A	N25 (NNNNNNNNNNNNNNNNNNNNNNNNN)	pRcap