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Preparation method of quantitative standard substance for high-throughput sequencing library of illumina platform
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A quantitative standard and sequencing library technology, applied in the direction of microbiological-based methods, biochemical equipment and methods, microbiological determination/inspection, etc., to achieve accurate quantification and high method accuracy
Active Publication Date: 2020-11-20
NAT INST OF METROLOGY CHINA
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[0004] At present, there is no preparation method for quantitative reference materials for high-throughput sequencing libraries in China
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Embodiment 1
[0046] Preparation and determination of quantitative reference materials for high-throughput sequencing libraries on the Illumina platform
[0047]1. Materials and methods:
[0048] 1. Escherichia coli (E.coli) genomic DNA extraction
[0049] After streak culture of Escherichia coli strain BL21(DE3), pick a single colony for overnight liquid culture, centrifuge at 5000g, discard the supernatant, collect the bacteria, and use Kangwei Century’s general genome extraction kit (CW2298) for genome extraction .
[0050] When the tested sample is a human genome, it can be replaced with other non-human genomic DNA; when the tested sample is a microorganism, it can be replaced with other non-target microorganisms or human-derived genomic DNA.
[0051] 2. Random interrupt
[0052] The above-mentioned extracted and purified E.coli genomic DNA was randomly interrupted by the Covaris S220 instrument, and the processing conditions were: intensity: 5, time: 6min, sample volume: 100μL, DNA ...
Embodiment 2
[0090] Example 2 High-throughput sequencing library quantitative reference material applicability verification
[0091] 1. Materials and methods:
[0092] 1. Library Quantitative Reference Material
[0093] The 5-level library quantitative reference material was developed by the China Institute of Metrology.
[0094] 2. Library quantification kit
[0095] Library quantification kits were purchased from KAPA and NEB respectively.
[0096] 3. Measurement of standard substance linearity by fluorescent quantitative PCR
[0097] Roche480 fluorescent quantitative PCR instrument was used to measure the quantitative standards of KAPA Company and Qiagen Company respectively. 20 μL of PCR amplification system: 10 μL of 2×SYBR PCR mastermix, 0.4 μL of bidirectional primer (10 μM), 4 μL of DNA template, ddH 2 O 5.6 μL. PCR reaction program: 95°C for 5min, 95°C for 20s, 60°C for 40s, 45cycles, melting curve: 95°C for 5s, 65°C for 1min, continuous signal acquisition at 95°C.
[0098]...
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Abstract
The invention discloses a preparation method of a standard substance for quantifying an Illumina platform high-throughput sequencing library. The preparation method comprises the following steps: carrying out ultrasonic fragmentation on escherichia coli genome DNA (Deoxyribonucleic Acid); carrying out tail end repairing and connector connection, and carrying out magnetic bead selection and recycling to obtain a library quantifying standard substance; determining a quantity value through an established dyestuff digital PCR (Polymerase Chain Reaction) method. The preparation method of the standard substance, disclosed by the invention, takes fragmented escherichia coli genome DNA as the library quantifying standard substance and library construction of a sample in actual detection can be simulated better. A constant value method of the standard substance has high accuracy and does not depend on a DNA standard product; measurement results can trace the number of the substance, so that the reliability and the traceability of the measurement results are ensured. The standard substance can be used as a measurement standard and is used for accurately quantifying the DNA of the Illumina platform high-throughput sequencing library.
Description
technical field [0001] The invention relates to the field of gene detection, in particular to a method for preparing a quantitative standard substance of a high-throughput sequencing library. Background technique [0002] Gene sequencing technology and industry began with the Human Genome Project launched in 1990, which is recognized by the global biotechnology community as a basic technology that has a profound impact on human health and an industry with strong spillover benefits. Next generation DNA sequencing (NGS) with ultra-high sequencing throughput has promoted the rapid development of the gene industry at a speed exceeding Moore's Law. The accuracy of gene sequence measurement is directly related to people's judgments on life activities, such as cancer diagnosis and species identification. Since NGS can sequence hundreds of thousands to millions of DNA molecules at a time, it makes it convenient and easy to deeply sequence the genome of a species. [0003] At prese...
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