Unlock instant, AI-driven research and patent intelligence for your innovation.

Design method and application of capture probes based on next-generation sequencing

A design method and a technology for capturing probes, which are applied in biochemical equipment and methods, sequence analysis, microbial measurement/inspection, etc., can solve problems such as balance, affecting product detection performance, and inability to guarantee probe capture performance, so as to improve Uniformity, which is conducive to the effect of stable detection

Active Publication Date: 2021-06-15
上海思路迪医学检验所有限公司
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the existing technology adopts the probe tiling design scheme with a fixed number of layers to ensure that the number of probe coverage layers in different areas is consistent, it cannot guarantee the balanced capture performance of probes in different areas, which may affect the detection performance of the product

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Design method and application of capture probes based on next-generation sequencing
  • Design method and application of capture probes based on next-generation sequencing
  • Design method and application of capture probes based on next-generation sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Probe Design

[0040] Using the probe design scheme of the present invention, all exon regions of 110 tumor-related genes (Table 1) were selected as target regions, and the relationship between GC content in target regions and probe capture efficiency in tumor FFPE samples was evaluated and a simulated After fitting the curve ( figure 1 ), and design two sets of probes according to the fitting curve, one set is to tile the probes in the same layer (two layers) in each target exon region ( image 3 Zhongyuan scheme), the other is to dynamically design the number of probe layers in each exon region according to the GC content ( image 3 In the new scheme), the specific design scheme is to tile 4 layers of probes if the GC content of the target exon region is less than 40%, and tile 2 layers of probes if the GC content of the target exon region is greater than 40%. The length of the probes is 120bp, and the two sets of probes are used to capture the same tumor ...

Embodiment 2

[0043] Example 2 Library construction and sequencing of tissue samples

[0044] After obtaining the FFPE roll of the tissue sample, use the QIAamp DNA FFPE Tissur Kit (50) (product number: 56404) to extract the DNA in the FFPE sample, and then use the M220 ultrasonic breaker to break and screen the length Double-stranded DNA fragments around 200 bases are used for subsequent library capture. The IDT prism kit (xGEN PrismDNA Library Prep Kit) was used for library construction, and the specific operation procedure refers to the kit instruction. After the library construction is completed, the libraries of one to four samples are mixed, and the two sets of probes designed by the present invention are used to capture respectively, and the IDT (Integrated dna Technologies) capture kit (XGen Hybridization and wash kit). The captured library was sequenced using illumina NextSeq 550Dx.

Embodiment 3

[0045] Example 3 Bioinformatics Analysis

[0046] 1 Sequencing data preprocessing and quality control

[0047] After obtaining the sequencing data BCL file, use the bcl2fastq v2.19.0 software to convert the sequencing off-machine file (BCL format) into a sequence file (FASTQ format), and then use the fastp v0.20.0 software to perform quality control (QC) and filter ( filter) to remove low-quality sequences, and then use bwa v0.7.12 software to align high-quality clean sequences to the human reference genome (GRCh37), generate an alignment file (BAM format), and use sambamba v0.6.5 software for sorting ( sort) and index (index) and count the average sequencing depth information (depth) in the target region (that is, each exon region), and use the local Python script to calculate the GC content of the target region.

[0048] 2 Evaluate the probe capture efficiency of the new scheme of probe design

[0049] According to the GC content of the target exon region, the target regio...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a design method and application of a capture probe based on next-generation sequencing. In order to make the capture performance of all target areas not affected by the GC content of the area, the present invention adopts different probe design schemes in different GC content areas, and according to the dynamic relationship between the probe capture efficiency and the GC content of the probe coverage area Adjust the number of probe layers in different regions, increase the number of probe layers in areas with low GC content, and / or reduce the number of probe layers in areas with high GC content; and further adjust the overlap between adjacent probes length, thereby improving the uniformity of probe capture across the target region.

Description

technical field [0001] The present invention relates to the field of next-generation sequencing, in particular to a method for designing a capture probe based on next-generation sequencing and its application. Background technique [0002] Next generation sequencing (next generation sequence, NGS) is a common technique for detecting unknown mutations in tumor samples. Whole genome sequencing has disadvantages such as high cost, long cycle, and low effective data rate. Therefore, probe capture method is commonly used in clinical molecular diagnosis to detect targets. The clinical variation of the gene guides the medication. DNA probes are generally designed using the DNA sequence of the target region as a template, with a length of tens to hundreds of bases, and are sequenced by capturing the target gene sequence by completely or partially matching the DNA of the target region. The basic idea of ​​the current probe design method for nucleic acid detection products is as foll...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): G16B25/20G16B30/00C12Q1/6869
CPCC12Q1/6869G16B25/20G16B30/00C12Q2535/122
Inventor 韩志军曹鑫恺周红霞秦放王杰
Owner 上海思路迪医学检验所有限公司