Spatial indexing of genetic material and library preparation using hydrogel beads and flow cells

A technology of hydrogel beads, genetic material, applied in specific-purpose bioreactors/fermenters, libraries, chemical libraries, etc., can solve problems such as increasing the complexity of SBS methods, achieve simplified sequence reconstruction, improve efficiency, eliminate The effect of cumbersome barcode steps

Pending Publication Date: 2020-05-05
ILLUMINA INC
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AI Technical Summary

Problems solved by technology

Pre-sequencing steps, such as barcoding specific nucleic acid molecules, can simplify data analysis but also add complexity to SBS methods

Method used

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  • Spatial indexing of genetic material and library preparation using hydrogel beads and flow cells
  • Spatial indexing of genetic material and library preparation using hydrogel beads and flow cells
  • Spatial indexing of genetic material and library preparation using hydrogel beads and flow cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0305] Flow Cell Capture of Degradable Hydrogel Beads Holding Encapsulated Nucleic Acid Molecules

[0306] This example illustrates the preparation of hydrogel beads encapsulating nucleic acid molecules using a manual vortex and a microfluidic droplet generator.

[0307] The pore size of the hydrogel beads is chosen to allow diffusion of enzymes, chemicals, and smaller sized primers (300bps), allowing genomic DNA and resulting DNA libraries to remain in the gel beads during the process.

[0308] A 12% hydrogel solution was prepared from 40% acrylamide / bis 19:1 (BioRad #161-0144) diluted with deionized water. Prepare a 40% (w / v) acrylamide / N,N'bis(acryloyl)cystamine (BACy) (19:1) monomer stock solution (3.8 g acrylamide, 0.2 g BACy and 6 mL double distilled (dd) HO ). The mixture was brought to a final volume of 10 mL. To prepare the solution, dissolve acrylamide in 6 mL of ddHO and use the resulting solution to dissolve BACy. Dissolution of the monomer is endothermic, so ...

Embodiment 2

[0317] In-bead sequencing library preparation and flow cell seeding

[0318] This example illustrates the preparation of a sequencing library from genomic DNA held in hydrogel beads captured on a sequencing flow cell, and the subsequent seeding of the prepared nucleic acid library on the flow cell.

[0319] As described in Example 1, in the MiSeq TM Hydrogel beads containing the encapsulated genomic DNA fragments are captured on the surface of the flow cell. A solution containing enzymes and reagents for DNA library preparation from genomic DNA in beads captured on the sequencing flow cell was flowed onto the flow cell to prepare the DNA library in hydrogel beads ( Figure 4A with 4B ). The pore size of the captured hydrogel beads allows diffusion of enzymes, chemicals, and smaller sized primers (300bps). Mineral oil is then loaded onto the sequencing flow cell to fill the voids between the beads and surround the captured hydrogel beads containing the DNA library. Then th...

Embodiment 3

[0322] Sequencing Nucleic Acids Using Hydrogel Beads

[0323] This example presents the results of a sequencing assay using hydrogel beads to spatially index nucleic acid molecules on the surface of a flow cell.

[0324] Three different workflows were determined for sequencing long DNA molecules (see Figures 5A-5C ). Two different DNA fragment lengths (~100 kb and ~10 kb) were tested, along with an in-bead MDA step prior to sequencing library preparation to amplify genomic DNA in hydrogel beads.

[0325] Hydrogel beads containing encapsulated genomic DNA fragments of ~100 kb or ~10 kb were generated as described in Example 1 . For each fragment length, in-bead MDA was assessed prior to library generation.

[0326] Load the resulting hydrogel beads into the MiSeq TM Sequencing on a flow cell and using Nextera TM (Illumina, SanDiego, CA) reagents and protocol to generate sequencing libraries by tagging reactions. Mineral oil is then loaded into the flow cell to surround t...

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Abstract

Implementations of a method for seeding sequence libraries on a surface of a sequencing flow cell that allow for spatial segregation of the libraries on the surface are provided. The spatial segregation can be used to index sequence reads from individual sequencing libraries to increase efficiency of subsequent data analysis. In some examples, hydrogel beads containing encapsulated sequencing libraries are captured on a sequencing flow cell and degraded in the presence of a liquid diffusion barrier to allow for the spatial segregation and seeding of the sequencing libraries on the surface of the flow cell. Additionally, examples of systems, methods and compositions are provided relating to flow cell devices configured for nucleic acid library preparation and single cell sequencing. Some examples include flow cell devices having a hydrogel with genetic material disposed therein, and which is retained within the hydrogel during nucleic acid processing.

Description

[0001] related application [0002] 本专利申请要求于2018年4月26日提交的美国临时专利申请号62 / 663,129和2017年8月1日提交的美国临时专利申请号62 / 539,949的优先权,其全部内容通过引用合并于此。 Background technique [0003] 边合成边测序(SBS)技术提供高质量的测序数据。但是,SBS方法可能受序列读出长度的限制,因为在某些情况下,SBS测序读出的长度不超过300个核苷酸。SBS技术的短读长度可能涉及大量数据分析,以从SBS程序期间生成的多个重叠的短序列读出进行对齐和重建长核酸序列。预测序步骤(例如对特定核酸分子进行加条形码编码)可以简化数据分析,但也会增加SBS方法的复杂性。 Contents of the invention [0004] 本文提供了用于在测序流动池上播种由遗传物质(例如靶核酸分子或含有靶核酸分子的细胞或细胞裂解物)产生的测序文库的方法的示例,其允许在流动池上各个文库的空间隔离。空间隔离可用于索引来自各个测序文库的序列读出,以提高后续数据分析的效率。测序文库的这种“空间索引”允许简化从中产生测序文库的遗传物质的处理和序列重建。在其他改进中,所公开的方法可以减少并且在某些情况下甚至消除对用于识别与特定遗传物质有关的序列读出的繁琐条形码步骤的需要。本文所述的示例还增加了用于靶核酸分子测序的数据分辨率,并进一步简化了基因组的组装(例如对于新的生物),并提供了对罕见基因变异和靶核酸分子中突变共现的改进识别。 [0005] 在一些示例中,提供了一种方法,其包括在足以将水凝胶珠捕获在测序流动池的表面上的条件下,将可降解水凝胶珠加载到测序流动池上。 在一些示例中,可降解水凝胶珠容纳由包封的遗传物质制备的测序文库。 在一些示例中,可降解水凝胶珠容纳包封的遗传物质,并且该方法进一步包括在所捕获的可降解水凝胶珠中从遗传物质制备测序文库。 然后将包围所捕获的水凝胶珠的液体扩散屏障加载到测序流动池上,并在存在液体扩散屏障的情况下降解所捕获的水凝胶珠,以允许将测序文库转运和播种到测序流动池 on the surface. [0006] 遗传物质可以是例如靶核酸分子,例如基因组DNA(例如人基因组DNA),以及含有靶核酸分子的细胞和细胞裂解物。 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6874C12Q1/6869C12M1/34
CPCB01L3/502753B01L2300/0877B01L2300/069B01L2200/0652B01L2200/0668C12Q1/6806C40B40/06C12Q1/6874C12Q2563/149C12Q2565/629C12Q1/6869C12Q2565/514C12Q2523/319C12Q2527/125C12Q1/6813C12Q2600/156
Inventor T·K·库拉纳吴怡萱陈锡君F·戈尔佩-亚萨尔林彦佑V·波皮克E·B·耶格M·罗纳格希
Owner ILLUMINA INC
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