Ultra-low-frequency library building method for FFPE tissue samples

A tissue sample, ultra-low frequency technology, applied in biochemical equipment and methods, chemical libraries, combinatorial chemistry, etc., can solve problems such as inaccurate detection, DNA loss, and undetectable ultra-low frequency mutations

Inactive Publication Date: 2018-04-17
普瑞基准科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1. The amount of double-stranded DNA required to build a library is relatively large, so it cannot meet the needs of some clinical tissue samples, such as FFPE samples of punctured tissues, FFPE samples of 5-10 years, etc.;
[0005] 2. Due to the multi-step purification process in the process of building the library, some short or ultra-short DNA, single-stranded DNA, and severely damaged DNA are all lost, which undoubtedly makes the already precious samples worse, resulting in Some ultra-low frequency mutations cannot be detected;
[0006] 3. Due to the need to perform end repair, PCR enrichment, and computer sequencing on some severely degraded samples during the library construction process, there is a possibility of introducing technical mutations in these processes, resulting in inaccurate detection
[0008] 1. During the single-stranded library construction process, the connection between the single-stranded adapter and the single-stranded DNA has a preference, and the connection efficiency is relatively low, which still requires a large initial sample size for library construction;
[0009] 2. In the existing single-stranded DNA library construction, there are still common problems with double-stranded DNA library construction, that is, there are also mutations introduced by human manipulation that cannot be ruled out

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] An ultra-low frequency method for building a library of FFPE tissue samples, the steps of which are as follows:

[0051] Step 1, extraction of FFPE sample DNA;

[0052] Step 2. DNA fragmentation treatment: During the extraction process, the extracted FFPE DNA samples need to be divided into four types according to the degree of degradation shown by gel electrophoresis: no degradation, with a main band and the size of the main band is greater than 2K; mildly degraded, with Main band but the range of the main band is between 1K and 2K; Moderately degraded, no main band, the bands are diffuse, and the fragment range is between 100bp and 2kb; Severely degraded, no main band, the fragment size is less than 500bp; severely degraded samples , no need to interrupt, directly build the library, no degradation, mild degradation and moderate degradation need to be interrupted by ultrasound;

[0053] Step 3, dephosphorylation and denaturation of DNA: use the dephosphorylase FastAP ...

Embodiment 2

[0061] Step 1. Obtaining the single-chain linker: synthesize two sequences according to the following sequence information:

[0062] Adapter sequence (A): 5'Pho-GCTACCCCAC-NNNNNNNNNNNN-AGATCGGAAG-XXXXXXXXXXXX-TEG-Biotin;

[0063] Sequence (A) is a linker sequence, which consists of 5 parts, including a 10nt base sequence region, a 12nt UID region, a 10nt illumina linker region, 10 C3-Spacer regions and a biotin region combined with streptomycin;

[0064] Splinter sequence (B): 3'NNNNNN-CGATGGGGTG5';

[0065] Sequence (B) is a Splinter sequence, consisting of 2 parts, including a 6nt random sequence region and a 10nt complementary region,

[0066] Purification of Adaptor (A): The total reaction system is 20 ul: 2 ul of 10 µM A sequence, 16 ul 1 x T4 RNA ligase buffer, 1 ul Klenow fragment of E.coli DNA polymerase I, 1 ul T4 long nucleoside Acid kinase; the reaction conditions are 37°C, 20min, 95°C, 2min.

[0067] Splinter sequence (B) purification: the total reaction system ...

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Abstract

The invention discloses an ultra-low-frequency library building method for FFPE tissue samples. The ultra-low-frequency library building method for FFPE tissue samples comprises the following steps: DNA extraction of FFPE samples; DNA interruption; dephosphorylation and DNA denaturation; adding of biotin UID single-chain joints; capturing and elution of streptomycin magnetic beads; adding of double-chain second joints and elution of a library; adding of a second joint at the other end of a target DNA molecule by T4DNA ligase, and removal of excessive and residual joints by washing; LM-PCR amplification and introduction of barcode; and purification of a PCR amplification product by magnetic beads. Library building, capturing and sequencing are realized for some severely degraded FFPE DNA micro-samples, UID label joints are introduced before beginning of library building, and thus, preconditions are provided for follow-up exclusion of technical mutations introduced in library building, capturing and computerized sequencing process.

Description

technical field [0001] The invention relates to an ultra-low frequency library building method for FFPE tissue samples, which mainly solves the problem of library building and sequencing of FFPE samples in the NGS-based sequencing process. Background technique [0002] At present, there are two main types of database construction methods for FFPE samples: [0003] The first category is the method of double-stranded DNA library construction. The shortcomings of this method are mainly manifested in the following aspects: [0004] 1. The amount of double-stranded DNA required to build a library is large, so it cannot meet the needs of some clinical tissue samples, such as FFPE samples of punctured tissues, FFPE samples of 5-10 years, etc.; [0005] 2. Due to the multi-step purification process in the process of building the library, some short or ultra-short DNA, single-stranded DNA, and severely damaged DNA are all lost, which undoubtedly makes the already precious samples wo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2525/191C12Q2531/113
Inventor 张仕坚严建龙
Owner 普瑞基准科技(北京)有限公司
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