Spinal muscular atrophy related gene copy number detecting kit and method based on gene trapping and second-generation sequencing technique

A next-generation sequencing technology, a technology for spinal muscular atrophy, applied in the field of biomedicine, can solve the problems of uneven sequencing depth, poor stability, and high error rate, achieve high throughput, improve detection sensitivity, and good robustness Effect

Active Publication Date: 2017-06-13
上海序祯达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a spinal muscular atrophy-related gene copy number detection kit based on gene capture and next-generation sequencing technology, which is used to solve the existing method of uneven sequencing depth, high error rate, stable Problems such as poor performance and low flux

Method used

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  • Spinal muscular atrophy related gene copy number detecting kit and method based on gene trapping and second-generation sequencing technique
  • Spinal muscular atrophy related gene copy number detecting kit and method based on gene trapping and second-generation sequencing technique
  • Spinal muscular atrophy related gene copy number detecting kit and method based on gene trapping and second-generation sequencing technique

Examples

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Embodiment 1

[0062] Example 1 Sequencing of samples (obtaining sequence information of polynucleotide fragments from samples)

[0063] Samples are sequenced through the following steps:

[0064] 1) DNA extraction and fragmentation. The sample used can be either blood or saliva. DNA is extracted and purified, and the extracted DNA is interrupted by ultrasonic waves, the ends are filled and phosphorylated, and adapters are added on both sides.

[0065] 2) Target area capture. In this method, a specially designed capture probe is used to capture the target region, magnetic beads are separated and enriched, PCR is amplified, and a sequencing library is constructed. Here, the target region includes the exons of SMN1 / 2 and some other genes and bases within a certain range upstream and downstream. Wherein, the target capture region and the corresponding probe sequence of SMN1 / 2 exon 7 are as described in Table 1, figure 1 The exon 7 of the SMN1 gene and the SMN2 gene and the three different ...

Embodiment 2

[0076] Example 2 Estimating the copy number of genes related to spinal muscular atrophy through data analysis.

[0077] The data analysis of the present invention involves the following steps:

[0078] Step A, aligning the sequencing reads (reads) to the human reference genome (hg19) using alignment software (BWA software, version: 0.7.12).

[0079] Step B, removing repetitive sequences. Use Picard (a basic sequence processing tool, version 2.8) to remove repetitive sequences (PCR duplicate reads) generated during PCR amplification.

[0080] Step C, removing reads whose alignment results are not credible. Here, the unreliable alignment result means that because SMN1 and SMN2 are highly homologous, although the alignment software assigns a read to exon 7 of one of the genes, in fact the read can be assigned to SMN1 , can also be assigned to SMN2. For the reads assigned to SMN1 / 2 exon 7, the present invention eliminated those reads that did not cover any of the sites describ...

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Abstract

The invention discloses a spinal muscular atrophy related gene copy number detecting kit and method based on a gene trapping and second-generation sequencing technique. The kit comprises capture probes, wherein the capture probes include four main probes and related control probes. The four main probes are the probe for capturing an SMN2 gene exon 7 and shown as SEQ ID NO:1, the probe for capturing an SMN2 gene exon 7 and shown as SEQ ID NO:2 the probe for capturing an SMN1 gene exon 7 and shown as SEQ ID NO:3 and the probe for capturing an SMN1 gene exon 7 and shown as SEQ ID NO:4, the capture probes conduct capture and sequencing on a target area, and the copy number of SMN1 and SMN2 gene exons 7 is further estimated through the data analysis method. The problem that an existing method is not uniform in sequencing depth, higher in error rate, poor in stability, smaller in flux and the like is solved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a detection kit, in particular to a detection kit for the copy number of spinal muscular atrophy-related genes based on gene capture and next-generation sequencing technology. In addition, the present invention also relates to a method for detecting the copy number of spinal muscular atrophy-related genes based on gene capture and next-generation sequencing technology. The method employs region-of-target capture and next-generation sequencing, and involves statistical analysis of the data for estimating the copy number of SMA-related genes. Background technique [0002] Spinal muscular atrophy (SMA) is a kind of disease caused by the degeneration of motor neurons in the anterior horn of the spinal cord leading to muscle atrophy. According to the incidence and clinical manifestations of patients, SMA can be divided into four types: type I (severe type), type II (intermediate type), typ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G06F19/24
CPCC12Q1/6869G16B40/00C12Q2535/122C12Q2565/519C12Q2537/16C12Q2537/165
Inventor 孟鑫彭建龙戴珩
Owner 上海序祯达生物科技有限公司
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