WO2024233135A1

WO2024233135A1 - Long-read sequencing of diverse dna libraries through barcode-labeled short reads

Info

Publication number: WO2024233135A1
Application number: PCT/US2024/026365
Authority: WO
Inventors: Ana CARNEIRO; David V. Schaffer; Danqing Zhu
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2023-05-05
Filing date: 2024-04-26
Publication date: 2024-11-14
Anticipated expiration: 2025-11-05

Abstract

Barcode-labeled short reads (BLaSR) is used for long-read sequencing of diverse DNA libraries, by dual transformation of AAV and Tn5-barcode libraries into E.coli; isolation or extraction of Tn5-inserted AAV cap sequences; next-generation sequencing and grouping of reads with the same barcode; an assembly of cap contigs based on Tn5 barcode to provide accurately-sequenced cap libraries.

Description

Long-Read Sequencing of Diverse DNA Libraries through Barcode-Labeled Short Reads

[001] Introduction

[002] A downfall of short-read sequencing technologies - the inability to sequence long stretches of DNA - has been addressed by newer, long-read technologies, such as Oxford Nanopore and PacBio SMRT Sequencing. These sequencers can characterize longer read lengths ranging from a few thousand to several thousands of base pairs. However, long-read sequencing techniques, particularly single-molecular long-read technologies, suffer from high error rates, ranging up to 15%, which cannot capture single-nucleotide variations in highly diverse DNA libraries. In addition, current methodologies are often labor-intensive and produce highly variable results. There is a need for accurate profiling of highly-di verse DNA libraries with minimal error that existing long-read sequencing technologies cannot achieve.

[003] The ability to deliver genetic material into target cells to correct or supplement defective genes is an efficient method of treating or preventing certain diseases. Over the past decade, AAV has emerged as a safe and highly effective vehicle for delivering DNA for clinical gene therapy. The ability to promote high expression of the therapeutic and lack of pathogenicity make AAVs an attractive option for gene therapy. AAVs have already seen success in early clinical trials. Recent approvals of AAV therapies by the US Food and Drug Administration (FDA), such as Luxtuma and Zolgesma, have highlighted the potential of AAV gene therapies.

[004] Issues still arise over the efficiency and specificity of AAV gene delivery, as well as the possible immune responses from naturally occurring AAV vectors. With over 60% of the human population already exposed to naturally occurring AAVs, the existence of antibodies against AAV can significantly limit the effectiveness of any AAV-based treatment or re-administration of the therapeutic. To overcome this, engineering the AAV capsid, or virus shell, is necessary to improve both improve its delivery efficiency and prevent recognition of our body’s antibodies. [005] Directed evolution is a high-throughput approach for creating and identifying novel AAV variants with enhanced properties, such as infectivity, targeted delivery, tissue spread, or immune evasion. The ability to deeply sequence these libraries offers insight into the evolutionary process, as well enable earlier identification of effective variants; however, accurate characterization of these diversified AAV libraries is not possible due to limitations in current sequencing technologies. Short read next-generation sequencing has a relatively low error rate and can capture variant mutations in which mutations are restricted to a specific region or within the read length, yet libraries diversified via methods such as error-prone PCR or random shuffling, in which diversity is spread through the full cap ORF, are unable to be correctly sequenced as individual clones. Long read sequencing offers this potential, but at substantially lower read depth and high error rates (1-15%).

[006] Relevant Literature includes:

[007] Wetmore et al. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio. 2015 May 12;6(3):e00306-15;

[008] Price, M.N., Wetmore, K.M., Waters, R.J. et al. Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 557, 503-509 (2018);

[009] Illumina. High-performance long-read assay enables contiguous data with N50 of 6-7 kb on existing Illumina platforms. 2023. Html

[010] Summary of the Invention

[Oil] In an aspect the invention provides a method of sequencing, comprising long-read sequencing of diverse DNA libraries through barcode-labeled short reads (BLaSR).

[012] In embodiments the invention:

[013] comprises the steps essentially as depicted in Fig. 1 ;

[014] is configured to sequence and profile a diverse AAV cap (e.g. ~2.2kb) library;

[015] is applied to mutagenized adeno-associated virus (AAV) cap libraries that have been diversified across the cap open reading frame;

[016] uses a randomly-barcoded transposon system, such as Tn5;

[017] harnesses the random and unbiased mechanism of the transposon to insert random barcodes into the AAV cap library such that each cap variant contains insertions with a unique, identifiable barcode, which provides the generation of barcode-linked next-generation sequencing short reads in which reads with the same barcode can be traced to the same AAV variant and thus be assembled into the longer cap variant sequence, thus generate de novo assemblies of the library;

[018] is configured to provide a robust pipeline to ensure highly efficient barcoded-Tn5 insertion into the AAV genome and have assembled thousands of AAV cap variants with an average 20-40x read depth;

[019] is configured to provide long-read sequencing methodology applied to other (non AAV cap) diverse DNA libraries that cannot otherwise be accommodated by next-generation sequencing;

[020] is configured for the assembly of diverse AAV cap sequences, comprising the steps of: [021] a) dual transformation of AAV and Tn5-barcode libraries into E.coli;

[022] b) isolation or extraction of Tn5 -inserted AAV cap sequences (plasmids); [023] c) enrichment of Tn5-containing cap sequences;

[024] d) next-generation sequencing (e.g. Illumina) and grouping of reads with the same barcode; and

[025] e) assembly of cap contigs based on Tn5 barcode to provide accurately- sequenced cap library;

[026] comprises a pipeline for generating Tn5-inserted cap sequences, comprising:

[027] a) dual electroporation of Tn5 and AAV libraries;

[028] b) culture of TN5 inserted AAV plasmids;

[029] c) electroporation of Tn5-inserted AAV plasmids; and

[030] d) enrichment of Tn5 inserted AAV plasmids.

[031] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[032] Brief Description of the Drawings

[033] Figs. 1 A-D: Development of Barcode-Labeled Short Reads (BLaSR) pipeline. A) Schematic for BLaSR process for the assembly of diverse AAV cap sequences. B) Schematic of experimental pipeline for generating Tn5-inserted cap sequences. C) Design of randomly- barcoded Tn5 plasmid library. D) Restriction enzyme digest confirming Tn5 insertion into the AAV cap gene.

[034] Figs. 2A-F : Assessment and optimization of Tn5 insertion bias. A) Restriction enzyme digest visualizing Tn5 insertion bias. B) NGS analysis of Read 1 coverage across AAV template before optimization of Tn5 bias. C) NGS analysis of Read 1 coverage across AAV template after optimization of Tn5 bias. D) NGS analysis of Read 2 coverage across AAV template before optimization of Tn5 bias. E) NGS analysis of Read 2 coverage across AAV template after optimization of Tn5. F) Locus sequence coverage of 5’ region before cap and cap region before and after optimization of Tn5 bias.

[035] Figs. 3A-C: Assembly of AAV cap sequences. A) Analysis of number of unique barcodes identified versus the number of sequences linked with each unique barcode. B) A subset of AAV assembled cap contigs, indicating the number of sequences used to generate the assembly versus the percentage of the full cap sequence assembled. C) Number of unique barcodes that have a particular number of sequences per barcode.

[036] Figs. 4A-B: Characterization of BLaSR. A) Error rate of the BLaSR methodology, as applied to known sequence AAV2 cap. B) Assembly of a small subset of AAV cap variants, demonstrating read depth across the AAV cap sequence. [037] Description of Particular Embodiments of the Invention

[038] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes. [039] The invention addresses the limitations of both long- and short-read sequencing methodologies to profile a diverse AAV cap (e.g. ~2.2kb) library. In aspects the invention provides a new synthetic long-read sequencing methodology we term Barcode-Labeled Short Reads (BLaSR) that can sequence a diverse AAV cap library. In aspects the invention provides mutagenized adeno-associated virus (AAV) cap libraries that have been diversified across the cap open reading frame. In embodiments the method uses a randomly-barcoded transposon system, such as Tn5. The Tn5 transposon is used in this methodology due to its ability to insert randomly and is the standard for non-biased insertion. Other types of transposons that demonstrate insertion biases can also readily be engineered with this random-barcode design and utilized in the BLaSR methodology.

[040] We harness the random and unbiased mechanism of the transposon to insert random barcodes into the AAV cap library such that each cap variant contains insertions with a unique, identifiable barcode. This allows for the generation of barcode-linked next-generation sequencing short reads in which reads with the same barcode can be traced to the same AAV variant and thus be assembled into the longer cap variant sequence, thus generate de novo assemblies of our library. We have engineered a robust pipeline to ensure highly efficient barcoded-Tn5 insertion into the AAV genome and have assembled thousands of AAV cap variants with an average 20-40x read depth. Optimization provides considerably larger numbers of sequenced cap variants per run.

[041] Use of BLaSR is not limited to AAV cap libraries; rather, this long-read sequencing methodology can be applied to other diverse DNA libraries that cannot otherwise be accommodated by next-generation sequencing. BLaSR can be applied to other regions of the AAV genome, such as AAV rep libraries. On a much broader level, BLaSR is applicable to any diversified DNA that falls outside the sequencing length of short-read technologies. Such libraries include other types of viral libraries, genomic libraries, and cDNA libraries. [042] The invention has broad applicability for a range of uses. The BLaSR methodology can be used to assemble de novo sequences using long reads, detect structural variants, sequence through entire genes, and more. Our tagmentation protocol can be used to label short reads for the de novo assembly of diverse DNA libraries. BLaSR can be used for long-read sequencing services, particularly those that focus on synthetic long-reads.

[043] We have engineered a randomly-barcoded Tn5 transposon to efficiently insert into DNA. The vector comprise a randomly-barcoded Tn5 transposon comprising inverted sequences flanking a selection marker and a random barcode, and a transposase. Our synthetic long-read methodology (BLaSR) addresses the pitfalls of current sequencing techniques. Here we have applied BLaSR to mutagenized adeno-associated virus (AAV) cap libraries that have been diversified across the entire cap open reading frame.

Claims

1 . A method of sequencing, comprising long-read sequencing of diverse DNA libraries through barcode-labeled short reads (BLaSR).

2. The method of claim 1, comprising the steps essentially as depicted in Fig. 1.

3. The method of claim 1, configured to sequence and profile a diverse AAV cap (e.g. ~2.2kb) library.

4. The method of claim 1, wherein the method is applied to mutagenized adeno-associated virus (AAV) cap libraries that have been diversified across the cap open reading frame.

5. The method of claim 1, that uses a randomly-barcoded transposon system, such as Tn5.

6. The method of claim 1 , that harnesses the random and unbiased mechanism of the transposon to insert random barcodes into the AAV cap library such that each cap variant contains insertions with a unique, identifiable barcode, which provides the generation of barcode-linked next-generation sequencing short reads in which reads with the same barcode can be traced to the same AAV variant and thus be assembled into the longer cap variant sequence, thus generate de novo assemblies of the library.

7. The method of claim 1, configured to provide a robust pipeline to ensure highly efficient barcoded-Tn5 insertion into the AAV genome and have assembled thousands of AAV cap variants with an average 20-40x read depth.

8. The method of claim 1, configured to provide long-read sequencing methodology applied to other (non AAV cap) diverse DNA libraries that cannot otherwise be accommodated by nextgeneration sequencing.

9. The method of claim 1, configured for the assembly of diverse AAV cap sequences, comprising the steps of: a) dual transformation of AAV and Tn5-barcode libraries into E.coli; b. isolation or extraction of Tn5-inserted AAV cap sequences (plasmids); c) enrichment of Tn5-containing cap sequences; d) next-generation sequencing and grouping of reads with the same barcode; and e) assembly of cap contigs based on Tn5 barcode to provide accurately- sequenced cap library.

10. The method of any of claims 1-9, comprising a pipeline for generating Tn5-inserted cap sequences, comprising: a) dual electroporation of Tn5 and AAV libraries; b) culture of TN5 inserted AAV plasmids; c) electroporation of Tn5-inserted AAV plasmids; and d) enrichment of Tn5 inserted AAV plasmids.