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Linker for preparing MGI/BGI platform NGS library and application of linker

A library and platform technology, applied in the field of next-generation sequencing, MGI/BGI platform NGS library preparation adapters, can solve the problems of analysis and judgment affecting the ligation effect, low yield of library construction, etc., to facilitate analysis and evaluation, library construction The effect of high yield and low preparation cost

Pending Publication Date: 2020-11-24
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with other types of adapters, the standard ligation products of this type of adapter are more diverse; including unligated standards, fragments with adapter monomers at one end, adapter monomers at both ends, or adapter two at one end. Fragments of polymers, fragments with adapter monomers and adapter dimers at both ends, and fragments with adapter dimers at both ends, in addition to the above fragments, there are other miscellaneous bands, which affect the analysis and judgment of the connection effect
Additionally, this bubble adapter has a low yield for library preparation compared to Illumina adapters

Method used

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  • Linker for preparing MGI/BGI platform NGS library and application of linker
  • Linker for preparing MGI/BGI platform NGS library and application of linker
  • Linker for preparing MGI/BGI platform NGS library and application of linker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Design of novel adapters and corresponding amplification primers

[0040] The adapters designed for MGI / BGI platform NGS library preparation (hereinafter referred to as "new adapters") and their amplification primer sequences are shown in Table 1:

[0041] Table 1

[0042]

[0043] *The underline is the complementary sequence of the barcord sequence on the BGISEQ / MGISEQ platform.

[0044] The corresponding bubble adapter and amplification primer sequences are shown in Table 2:

[0045] Table 2

[0046]

[0047]

[0048] *The underline is the complementary sequence of the barcord sequence on the BGISEQ / MGISEQ platform.

Embodiment 2

[0049] Example 2 Preparation of novel joints and connection efficiency analysis

[0050] 1. Preparation of new joints:

[0051] The synthesized new linker sequences (HD Bottom 1 and HD Bottom 2) were dissolved in 1×TEN buffer to 100 μM, mixed at equal concentrations, and annealed (25°C, 2 hours) to form a new linker; then diluted with TE buffer to 10 μM, consistent with the concentration of the bubble joint.

[0052] 2. Connection efficiency analysis:

[0053] In this embodiment, the 300bp standard fragment is the test template, and the input amount of the sample is 100ng.

[0054] Ultima DNA Library Prep Kit for MGI was used to repair the end of the sample / add A (60uL system, reaction conditions: 30°C for 20min, 72°C for 20min, 4°C∞).

[0055] Adapter ligation: Use Fast T4 DNA ligase to connect two NGS adapters respectively (total system-100uL, reaction conditions: 20°C for 15min, 4°C∞); after the ligation reaction, use HieffNGS TMDNA Selection Beads magnetic beads were u...

Embodiment 3

[0062] Example 3 Two kinds of NGS joint library construction test

[0063] In this example, calf thymus (calfthymus) gDNA was used as a sample, and the Ultima DNALibrary Prep Kit for and OnePot TM II DNA Library Prep Kit for Library construction kit, compare the library output of two NGS connectors, see the results Figure 5 and Figure 6 .

[0064] The results show:

[0065] A. Ultima DNA Library Prep Kit for For the library construction kit, when the input amount is 2ng, the output of the new adapter library can reach 1.6μg, while the bubble adapter is only 0.95μg, a difference of 1.65 times; the input amount of 50ng also has a difference of 1.31 times.

[0066] B. One Pot TM II DNA Library Prep Kit for For the kit, under the same library construction conditions, the input amount is 10ng and 50ng, and the output of the new linker library is 2.44 and 139 times that of the bubble linker, respectively.

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Abstract

The invention discloses a linker for preparing an MGI / BGI platform NGS library. The linker is formed by partial complementary pairing of a DNA single chain with a sequence shown in SEQ NO. 1 and a DNAsingle chain with a sequence shown in SEQ NO. 2. The invention also discloses an amplification primer combination corresponding to the linker. The amplification primer combination comprises an upstream primer with a sequence shown in SEQ NO. 3 and a downstream primer with a sequence shown in SEQ NO. 4. Compared with the prior art, the novel linker has the advantages of high effective connection efficiency, high library building yield, low preparation cost and high discrimination of linker connection product fragments, and is convenient for analysis and evaluation of connection effects.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to next-generation sequencing technology, in particular to a linker used for preparing NGS library of MGI / BGI platform and its application. Background technique [0002] NGS connector is an essential component in next-generation sequencing, and it is the bridge connecting the DNA fragment to be tested and Flow-cell. The linker is essentially a short nucleic acid sequence, basically including Read 1 sequencing primer binding sequence, Read2 sequencing primer binding sequence, index and index sequencing primer binding sequence. Currently, commonly used adapters on the Illumina sequencing platform market include Y-type adapters-Illumina and U-type adapters-NEB. For the independently developed MGISEQ platform, MGI has developed its own MGI connector - a single-ended index connector. Because its shape is similar to foam, it is also called a bubble-shaped adapter (bubble-shaped Adapter). Its st...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/11C40B50/06
CPCC12Q1/6806C40B50/06C12Q2525/191C12Q2531/113C12Q2563/143C12Q2563/149
Inventor 李真真王家庭曹振宋东亮周其好
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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