Capture probes for large genomic rearrangement detection based on capture sequencing, kit and detection method

Abstract

The invention discloses a large genomic rearrangement (LGR) detection method based on capture sequencing. The capture probes of the invention comprise exon probes and single nucleotide polymorphism (SNP) probes. The exon probes comprise at least three probes aiming at each exon of the gene and 60bp regions at two ends of the exon, and can achieve 2X complete coverage for the exon and the 60bp regions at two ends of the exon. The SNP probes comprise SNP loci with the population occurrence frequency of 30%-70% in the gene intron region and the upstream and downstream within the 5000bp ranges ofthe gene. Meanwhile, the invention further provides an algorithm formed by integrating a reads depth algorithm module, an allelic imbalance algorithm module and a discordant sequence algorithm module,and the algorithm is used for carrying out large genomic rearrangement analysis on the NGS sequencing data. By adoption of the method, the LGR can be effectively detected, the requirement for the purity of a nucleic acid sample is lowered, false positive caused by single-base variation is reduced, and high detection performance is achieved for exon amplification.

Description

technical field [0001] The invention relates to a capture probe, a reagent kit and a detection method for large fragment rearrangement detection based on capture sequencing. Background technique [0002] Large genomic rearrangement (LGR) refers to the fragmentation and recombination of large fragments of DNA in the genome, which will lead to functional changes of genes in rearranged regions, including exon deletion (deletion) and amplification (duplication) . LGR is an important class of pathogenic mutations, and its accurate detection is crucial for clinical practice. [0003] Currently in clinical practice, multiple ligation-dependent probe amplification (MLPA) is the most widely used method for detecting large fragment genome rearrangements of BRCA1 / 2. MLPA has three main drawbacks: 1. Higher requirements for the purity of nucleic acid samples: Alcohol, metal ions, phenol and Trizol will all affect the detection performance of MLPA; 2. When a single base variation (SNV)...

AI Technical Summary

Problems solved by technology

This type of method is limited by the number of amplicons, and only 1 or 2 amplicons can be designed for each exon, so that the data sampling points for each exon are only 1 or 2

However, after the samples go through the steps of collection, transportation, nucleic acid extraction, experimental reaction, etc., the noise level accumulated in each link is different under different conditions, which makes the noise level of the experimental data very different, resulting in some samples that cannot meet the requirements of large fragment rearrangement detection. Require

In addition, when there are SNVs in the probe or primer region, false positives will be caused due to the decrease of amplification efficiency, especially the detection performance of single exon deletion or amplification is not enough

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