摘要
目的短串联重复序列(short tandem repeat,STR)遗传标记在人类个体识别、亲缘关系分析、动植物物种鉴定、农作物品种登记及杂交种纯度鉴定、临床疾病诊断等领域应用广泛。本团队在利用Illumina二代测序平台进行法医STR基因座靶向测序时,发现部分基因座正反向测序深度不平衡的现象,影响测序数据质量。本文通过研究不同STR基因座的重复区和扩增子区域中碱基含量与测序深度的关系,探究该现象的原因。方法利用STRSeqTyper122二代测序STR分型试剂盒对70份无关个体的DNA样本进行STR基因座复合扩增和文库构建,使用Mi Seq FGx平台进行测序,下机数据使用ForensicTyper软件进行分型和测序深度统计,随后分析测序深度与重复区和扩增子区域中碱基含量之间的关系。使用同样方法对前期发表的Ion PGM^(TM)平台STR测序数据进行分析。结果分析117个STR基因座的测序深度发现,基因座正向测序深度占总测序深度的比例与扩增子区域的TC碱基占比紧密相关:扩增子中TC比例低于40%时,随着TC含量降低,正向测序深度占比有明显的升高趋势;扩增子中TC比例高于60%时,随着TC含量升高,正向测序深度占比有明显下降趋势。同样分析Ion Torrent平台70份样本的32个STR测序数据,未观察到类似现象。结论MiSeq FGx平台在测序STR基因座时存在链偏好性,正反向测序深度与碱基TC比例密切相关。以上研究可为二代测序STR基因座选择、引物和扩增子设计、数据分析与解读提供重要参考,服务临床医学、法医学、植物学等领域应用。
Objective:Short tandem repeat(STR)genetic markers are widely used in human individual identification,kinship analysis,animal and plant species identification,crop variety registration,hybrid purity identification,clinical disease diagnosis,and other fields.When using the Illumina next generation sequencing platform for targeted sequencing of forensic STR loci,the authors found that the depth of forward and reverse sequencing of some loci were imbalanced,which affected the quality of sequencing data.In this paper,the cause of this phenomenon was investigated by studying the relationship between sequencing depths and base content in repeat regions and amplicons of STRs.Methods:The STRSeqTyper122 STR Genotyping Kit was used for multiplex amplification and library preparations.DNA samples from 70 unrelated individuals were sequenced on a MiSeq FGx platform.The ForensicTyper software was used for STR allele calling and sequencing depth statistics.Then the correlation between sequencing depth and base content in repeat regions and amplicons were analyzed.The same method was used to analyze some previously published STR sequencing data on the Ion PGM^(™)platform.Results:Analysis of the sequencing depth of 117 STR loci revealed that the proportion of forward sequencing depth to the total sequencing depth was closely related to the proportion of TC bases in the amplicon region.When the TC proportions in STR amplicons were less than 40%,the proportion of forward sequencing reads increased significantly as the TC content decreased.When the TC proportions in STR amplicons were higher than 60%,the proportion of forward sequencing reads decreased significantly as the TC content increased.Ion Torrent sequencing data of 32 STR loci for 70 unrelated individuals were analyzed similarly,but no obvious correlation was observed.Conclusion:The MiSeq FGx platform exhibited strand preference phenomenons when sequencing STRs.The depth of forward and reverse sequencing were closely related to TC ratios.The above research can provide important reference for STR locus selection,primer and amplicon design,data analysis and interpretation based on next-generation sequencing,and serve in clinical medicine,forensic science,botany and other application fields.
作者
彭加金
郭立亮
吴浩
张驰
赵杰
康克莱
季安全
王乐
PENG Jiajin;GUO Liliang;WU Hao;ZHANG Chi;ZHAO Jie;KANG Kelai;JI Anquan;WANG Le(School of Forensic Medicine,Kunming Medical University,Kunming 650500,China;Key Laboratory of Forensic Genetics of Ministry of Public Security,Institute of Forensic Science,Ministry of Public Security,Beijing 100038,China)
出处
《生命科学仪器》
2022年第5期39-45,共7页
Life Science Instruments
基金
公安部科技强警基础工作专项项目(2019GABJC15)
中央级公益性科研院所基本科研业务费项目(2019JB009)资助
关键词
测序深度
扩增子
二代测序
短串联重复序列
TC比例
Sequencing depth
Amplicon
Next generation sequencing
Short tandem repeats
TC ratio
