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Surface concatemerization of templates

a surface concatenation and template technology, applied in the field of surface concatenation of templates, can solve the problems of limiting read length, increasing the burden on the robustness of sequencing biochemistry, and daunting task of cataloguing human genetic variation and correlating this variation with susceptibility to disease, etc., to achieve the effect of improving densities, improving read quality, and prolonging read length

Inactive Publication Date: 2018-04-12
ILLUMINA CAMBRIDGE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The methods and products in this patent make it possible to amplify DNA sequences on surfaces at higher density. This allows for the creation of brighter clusters that have better quality reads, support for longer read lengths, faster sequencing times, and increased system robustness.

Problems solved by technology

The task of cataloguing human genetic variation and correlating this variation with susceptibility to disease is daunting and expensive.
In contrast, analysis of clonal amplifications requires near quantitative completion of each sequencing cycle, otherwise background noise progressively grows with each ensuing cycle severely limiting read length.
As such, clonal analysis places a bigger burden on the robustness of the sequencing biochemistry and may potentially limit read lengths.

Method used

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  • Surface concatemerization of templates
  • Surface concatemerization of templates
  • Surface concatemerization of templates

Examples

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example 1

[0075]This example describes solid phase amplification according to one embodiment, as illustrated in FIG. 1. A standard universal PhiX or CT418 library containing P5 and P7′ regions was used to attach the additional P5′ at the 5′ end of the already prepared library. This was accomplished by performing additional 18 PCR cycles. The libraries were diluted to 200 pM final and amplified using either standard primer or P7-P5′ primer. Each 50 ul PCR reaction contained 22 μl of H2O, 25 μl of 2× Phusion Mastermix (NEB), 1 μl each of the appropriate primer and DNA. After PCR, the resulting library concentrations were determined by the Nanodrop (Thermo Scientific) and diluted to 10 nM in buffer EB (QIAGEN)+0.05% Tween20. A flowcell was prepared by grafting HEG primers (lanes 1-4) or standard PE primers (lanes 5-8) using the protocol described in U.S. Pat. Nos. 8,536,477, 8,715,966, and U.S. Patent Application Pub. 2008 / 0280773, the content of each of which is incorporated by reference herein...

example 2

[0078]This example describes solid phase amplification according to an embodiment, as illustrated in FIG. 3. The experiments reported in FIG. 6 show the results from concatemerization of 20T / A linkers on the P5 / P7 grafting primers (referred as 20T-P7 / 20A-P5) instead of attaching the concatamer facilitating primers on to the library during PCR process. In this experiment, the flowcell was grafted with 20A / T Paired-end P5 / P7 primers alongside standard primers. The standard grafting protocol as described in incorporated materials of U.S. Pat. Nos. 8,536,477, 8,715,966, and U.S. Patent Application Pub. 2008 / 0280773 was followed and this grafted flowcell was used for cluster amplification on a cBOT clonal amplification system (Illumina Cat # SY-301-2002) using the TruSeq cluster generation kit for Genome Analyzer (Illumina) as per manufacturer's recommended protocol. CT418 libraries were used as template; amplified 105 cycles at 60° C. The flowcell was stained with SYBR Green and imaged ...

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Abstract

Presented herein are methods and compositions for concatenating template strands during the bridge amplification process. The methods are useful for surface amplification at improved densities. The methods and compositions provided herein enable creation of clusters which are brighter, but at the same densities as currently achieved using standard cluster amplification.

Description

BACKGROUND[0001]The task of cataloguing human genetic variation and correlating this variation with susceptibility to disease is daunting and expensive. A drastic reduction in this cost is imperative for advancing the understanding of health and disease. A reduction in sequencing costs will require a number of technical advances in the field. Technical advances that could reduce the cost of genome analysis include: (1) library generation; (2) highly-parallel clonal amplification and analysis; (3) development of robust cycle sequencing biochemistry; (4) development of ultrafast imaging technology; and (5) development of algorithms for sequence assembly from short reads.[0002]The creation of clonal amplifications in a highly-parallel manner is important for cost-effective sequencing. Sequencing is generally performed on clonal populations of DNA molecules traditionally prepared from plasmids grown from picking individual bacterial colonies. In the human genome project, each clone was ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/6874C12Q1/6806C12N15/10C12N15/64
CPCC12Q1/6874C12Q1/6806C12N15/10C12N15/64C12Q1/6853C12Q2525/155C12Q2533/101C12Q2535/122C12Q2565/537C12Q2565/543
Inventor BOUTELL, JONATHAN MARK
Owner ILLUMINA CAMBRIDGE LTD
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