利用基因组测序技术鉴定家蚕转基因位点方法的建立  

Establishment of A Genome Sequencing Method for Identification of Transgenic Sites in Silkworm

作　　者：周昂 蒋晶晶 左伟东[1] 陈欣[1] 童晓玲[1] 代方银[1] 李春林[1] ZHOU Ang;JIANG Jingjing;ZUO Weidong;CHEN Xin;TONG Xiaoling;DAI Fangyin;LI Chunlin(State Key Laboratory of Resource Insects,Southwest University/Key Laboratory of Sericultural Biology and Genetic Breeding,Ministry of Agriculture and Rural Affairs,Southwest University,Chongqing 400715,China)

机构地区：[1]西南大学资源昆虫高效养殖与利用全国重点实验室/西南大学农业农村部蚕桑生物学与遗传育种重点实验室,重庆400715

出　　处：《西南大学学报(自然科学版)》2025年第4期113-122,共10页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金项目(U20A2058,31830094);中央高校基本科研业务费自然科学项目(SWU-KQ22005)。

摘　　要：建立一种基于基因组测序技术高效鉴定家蚕转基因插入位点的方法,并分析测序深度对位点鉴定准确性的影响,从而为家蚕及其他物种利用基因组测序技术开展低成本、高通量的转基因位点鉴定提供参考。首先,采集两份家蚕转基因材料的蛹,进行全基因组测序,并经过质量控制筛选出高质量的测序后读段(reads)。以载体序列和家蚕基因组为参考,经两轮比对分析,得到覆盖基因组和载体的目标reads。利用目标reads上的基因组序列在家蚕泛基因组数据库中进行比对分析,进而获得插入位点的具体位置。随后,运用Seqtk软件对数据进行抽样,分析不同测序深度对位点鉴定准确性的影响。两份材料的基因组测序数据分别为9.76 G和11.18 G,质控后读段(clean reads)数量分别为32639026和37394695,测序深度约为20×。序列比对分析和聚合酶链式反应(PCR)实验验证结果显示:TransGene_1的插入位点位于1号染色体1899494 bp与1899495 bp之间;TransGene_2的插入位点位于21号染色体959510 bp与959515 bp之间,且插入位点均处于基因间区。利用两份转基因材料的基因组测序数据进行不同测序深度的模拟分析,发现转基因材料TransGene_1在测序深度为6×时就能够得到覆盖两侧断点的reads,实现精确定位,而TransGene_2则需要测序深度达到10×及以上才能实现精确定位。This study aims to establish a method for efficiently identifying transgenic insertion sites in silkworms through genome sequencing.It also analyzes how sequencing depth affects the accuracy of site identification,and provides a reference for low-cost,high-throughput transgenic site identification in silkworms and other species.Two silkworm transgenic materials were collected for whole genome resequencing.High-quality reads were selected through quality control.Target reads covering the genome and vector were obtained through two rounds of comparison analysis using the vector sequence and silkworm genome as references.The specific location of the insertion site was obtained by comparing the genome sequence in the target reads with the silkworm pan-genome database.Subsequently,we used the seqtk software to sample the data,and analyzed how different sequencing depths affected the accuracy of site identification.The genome sequencing data of the two materials were 9.76 G and 11.18 G,and the number of clean reads were 32639026 and 37394695,respectively,and the sequencing depth was about 20×.Sequence alignment analysis and PCR experimental verification results indicate that the insertion site of TransGene_1 is located between 1899494 bp and 1899495 bp on chromosome 1,while the insertion site of TransGene_2 is between 959510 bp and 959515 bp on chromosome 21.Both insertion sites were located in the intergenic region.Using the genome sequencing data of the two transgenic materials for simulation analysis under different sequencing depths,we found that the transgenic material TransGene_1 can obtain reads covering both breakpoints at a sequencing depth of 6×,achieving precise positioning,while TransGene_2 needs a sequencing depth of 10×or above to achieve precise positioning.

关 键 词：家蚕 转基因插入位点 基因组测序 测序深度 转基因鉴定 

分 类 号：S882[农业科学—特种经济动物饲养]