甲醛固定石蜡包埋标本二代测序全自动建库终文库浓度低的补救方法研究  

The remedial approaches for the low final library concentration in the fully automatic library construction of next generation sequencing for formaldehyde-fixed paraffin-embedded specimens

作　　者：赵盼盼 陈文丹 许洁[1] ZHAO Panpan;CHEN Wendan;XU Jie(Pathology Department,Guangdong Provincial People's Hospital,Guangzhou,Guangdong 510010,China)

机构地区：[1]广东省人民医院病理科,广东广州510010

出　　处：《医药前沿》2024年第30期35-37,41,共4页Journal of Frontiers of Medicine

摘　　要：目的:探讨甲醛固定石蜡包埋标本(FFPE)样本提取的DNA在自动建库中终文库浓度低的补救方法.方法:收集2022年8月—2023年3月广东省人民医院病理科初次自动建库得到的终文库浓度低于1 ng/μL的15例FFPE样本提取的DNA.应用人多基因突变检测试剂盒,选择泛癌种探针,DNA样本打断后运用Magnis BR建库,最后得到20μL终文库,对其浓度进行定量质控.每个样本分两组进行实验,A组标本DNA按照200 ng进行建库,B组标本DNA按100 ng进行建库.选取终文库浓度大于1 ng/μL样本进行测序,并对下机数据进行质控分析及结果验证.结果:A组实验所得预文库总量偏低,终文库浓度均低于1 ng/μL,不符合上机标准;B组实验所得终文库仅3例浓度低于1 ng/μL,其余12例高于1 ng/μL,片段分布在质控范围内(12/15,80%),两组建库所得终文库浓度比较,差异有统计学意义(P<0.05).对B组终文库质控合格的12例样本进行测序,结果显示9例各项测序数据质控合格,均符合生信分析的质控要求(9/12,75%),3例质控警戒,12例均检出具有明确或潜在临床意义突变,8例使用ARMS-PCR法或免疫组化对主要基因突变进行验证,结果相符.结论:适当调整建库时的DNA投入量,对初次建库中终文库浓度低的样本进行重新建库,可有效提升建库成功率和测序质量.该方法尤其适用于无法进行二次切片的FFPE样本,如穿刺小标本,从而提高二代测序检测的成功率,使更多患者受益.Objective To explore the remedy method of low final library concentration of DNA extracted from formaldehyde-fixed paraffin-embedded specimen(FFPE)in automatic database construction.Methods DNA was collected from 15 FFPE samples with final library concentration lower than 1 ng/μL,which were automatically constructed for the first time in the Department of Pathology of Guangdong Provincial People's Hospital from August 2022 to March 2023.A human polygenic mutation detection kit was used to select a pan-cancer probe.After DNA samples were broken,Magnis BR was used to build a library.Finally,a 20μL final library was obtained and its concentration was quantitatively controlled.Each sample was divided into two groups for the experiment.The DNA of group A was constructed according to 200 ng,and the DNA of group B was constructed according to 100 ng.The samples with the final library concentration greater than 1 ng/μL were selected for sequencing,and the quality control analysis and result verification were carried out on the disembarkation data.Results In group A,the total amount of pre-library was low,and the final library concentration was lower than 1 ng/μL,which did not meet the sequencing standard.In group B,the final library concentration was lower than 1 ng/μL in only 3 cases,and higher than 1 ng/μL in the remaining 12 cases,and the fragment distribution was within the quality control range(12/15,80%).The difference in the final library concentration between the two sets of libraries was statistically significant(P<0.05).Sequencing was performed on the 12 samples qualified for final library quality control in group B,and the results showed that 9 cases were qualified for all sequencing data quality control,the rest met the quality control requirements of biogenic analysis(9/12,75%),3 cases were alert for quality control.12 cases were detected with clear or potential clinical significance mutations,and 8 cases were verified by ARMS-PCR or immunohistochemistry for major gene mutations,and the results were co

关 键 词：二代测序 全自动建库 组织固定 甲醛 

分 类 号：R730[医药卫生—肿瘤]