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Construction of next generation sequencing (NGS) libraries using competitive strand displacement

A sequencing and dependency technology, applied in the field of NGS library construction, can solve problems, reduce reaction efficiency, consume flow cell space, etc., achieve excellent recovery, reduce the number of reads, and improve the detection effect

Active Publication Date: 2019-09-17
INTEGRATED DNA TECHNOLOGIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These dimers form clusters very efficiently, reducing reaction efficiency and consuming valuable space on the flow cell
This is especially problematic when dealing with ultra-low DNA inputs in the picogram range

Method used

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  • Construction of next generation sequencing (NGS) libraries using competitive strand displacement
  • Construction of next generation sequencing (NGS) libraries using competitive strand displacement
  • Construction of next generation sequencing (NGS) libraries using competitive strand displacement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This example demonstrates that, with the use of Ultra TM II library (New England BioLabs) or KapaHyper Prep (Kapa Biosystems) method obtained enhanced coverage depth obtained from NGS libraries prepared from high quality genomic DNA using the second embodiment of the CSD method. High-quality genomic DNA was extracted from the cell line NA12878 (ATCC). 1 or 10 ng of extracted DNA was sheared to an average size of 150 bp using sonication (Covaris S220), followed by end repair, which included phosphorylation of the 5' end with T4 polynucleotide kinase (PNK) for 30 minutes, followed by purification by 2.5X AMPure beads. For CSD processing, a P7 linker (SEQ ID NO: 11-16) hybridized to a truncated 3'ddN-blocked oligonucleotide (SEQ ID NO: 17) was ligated by blunt end ligation using mutant K159S T4 DNA ligase Ligation to end-repaired target fragments for 15 minutes followed by a 15 minute heat kill step. A P5 adapter (SEQ ID NO: 1 or SEQ ID NO: 2) was then ligated to the...

Embodiment 2

[0052] This example demonstrates that, with the use of Ultra TMThe depth of coverage obtained from NGS libraries prepared from circulating cell-free DNA (cfDNA) using the second embodiment of the CSD method was enhanced compared to the depth of coverage obtained with the II library. The “real” cfDNA samples were real cell-free DNA isolated from healthy individuals by Biochain, while the “mock” cfDNA samples were cell line genomic DNA (NA12878) sheared to 150 bp using Covaris S2. Libraries were prepared in triplicate from 1 ng of cfDNA using the CSD and NEB methods as described in Example 1. When compared to the NEB method, the average coverage depth of CSD was 3.6 times higher with the "real" cfDNA input and 2.3 times higher with the "mock" cfDNA input ( Figure 8C ).

Embodiment 3

[0054] This example demonstrates the enhanced depth of coverage obtained using the second embodiment of the CSD method from NGS libraries prepared from low quality genomic DNA extracted from FFPE samples compared to the depth of coverage obtained using NEB Ultra II libraries. FFPE samples were purchased from Asterand Bioscience. Libraries were prepared using as starting material 1 ng, 5 ng, or 10 ng of FFPE-derived genomic DNA sheared to an average size of 200 bp, as described above. When compared to the NEB method, the average coverage depth of CSD was 1.8-fold, 1.4-fold, and 1.3-fold higher with 1 ng, 5 ng, or 10 ng FFPE-derived genomic DNA, respectively ( Figure 8D ).

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PUM

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Abstract

The invention pertains to construction of next-generation DN A sequencing (NGS) libraries for whole genome sequencing, targeted resequencing, sequencing-based screening assays, rnetagenomics, or any other application requiring sample preparation for NGS.

Description

technical field [0001] The present invention relates to the construction of NGS libraries for whole genome sequencing, whole exome sequencing, targeted resequencing, sequencing-based screening assays, metagenomics, or any other application requiring Next Generation DNA Sequencing (NGS) sample preparation . Background technique [0002] Next-generation sequencing (NGS) has evolved into a very powerful tool in molecular biology, allowing rapid progress in fields such as genome identification, genetic testing, drug discovery, and disease diagnosis. As the technology continues to develop, the amount of nucleic acid that can be sequenced at one time continues to increase. This allows researchers to sequence larger samples and increase the number of reads per sample, enabling detection of small sequence variations in said samples. [0003] As the volume and complexity of NGS processing increases, so does the rate of experimental error. While most of these errors occur during se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/53C12N15/09C12N15/10C12P19/34
CPCC12Q1/6806C12Y605/01001C12Q1/6855C12Q2521/501C12Q2525/191C12Q2563/179C12Q2525/15C12Q2525/155C12Q2525/186C12Q2535/122C12Q2537/1373C12Q2537/161C12Q2537/162C12Q1/6869C40B40/06
Inventor 扎卡里·茨威科郑俞米娜·贾罗斯陈采夫约瑟夫·瓦尔德
Owner INTEGRATED DNA TECHNOLOGIES
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