Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy.
Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy.
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DOI：
10.3325/cmj.2011.52.299
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发表时间：
2011-06
影响因子：
1.9
通讯作者：
O'Hanlon KA
中科院分区：
文献类型：
--
作者：
Holland MM;McQuillan MR;O'Hanlon KA
利用平行阵列焦磷酸测序对线粒体DNA （mtDNA）序列进行反卷积，并提供mtDNA异质性的高分辨率分析。使用454 GS Junior仪器分析了30例个体mtDNA控制区的高变段1 （HV1）。模拟混合物用于评估该系统对混合物进行反卷积的能力，并可靠地检测异质性，包括同一母系的5个家庭成员之间的异质性差异。扩增子测序是对聚合酶链反应（PCR）产物进行测序，这些产物由包含多重标识符（MID）和焦磷酸测序适配器的引物产生。使用NextGENe®软件进行数据分析。常染色体短串联重复序列（STR）位点（D18S51）和Y-STR位点（DYS389 I/II）与HV1的一部分同时进行分析，以说明多路复用可以包含法医感兴趣的不同标记。混合物，包括异质变异，可以常规检测到1:250的成分比（20个小变异拷贝，覆盖率为5000个序列），并且可以很容易地检测到1:100(0.1%)，扩大覆盖率。对D18S51、DYS389 I/II和HV1后半部分的扩增子序列进行了成功的分割和分析。常规反卷积mtDNA混合物至1:250水平的能力允许对mtDNA异质性进行高分辨率分析，并对来自同一母系的个体进行分化。焦磷酸测序方法导致均聚物序列的分辨率较差，并且PCR/测序伪影需要类似于STR口吃和光谱穿透的过滤机制。此外，从跳跃PCR的嵌合序列必须处理，使该方法的操作。
To use parallel array pyrosequencing to deconvolute mixtures of mitochondrial DNA (mtDNA) sequence and provide high resolution analysis of mtDNA heteroplasmy. The hypervariable segment 1 (HV1) of the mtDNA control region was analyzed from 30 individuals using the 454 GS Junior instrument. Mock mixtures were used to evaluate the system’s ability to deconvolute mixtures and to reliably detect heteroplasmy, including heteroplasmic differences between 5 family members of the same maternal lineage. Amplicon sequencing was performed on polymerase chain reaction (PCR) products generated with primers that included multiplex identifiers (MID) and adaptors for pyrosequencing. Data analysis was performed using NextGENe® software. The analysis of an autosomal short tandem repeat (STR) locus (D18S51) and a Y-STR locus (DYS389 I/II) was performed simultaneously with a portion of HV1 to illustrate that multiplexing can encompass different markers of forensic interest. Mixtures, including heteroplasmic variants, can be detected routinely down to a component ratio of 1:250 (20 minor variant copies with a coverage rate of 5000 sequences) and can be readily detected down to 1:1000 (0.1%) with expanded coverage. Amplicon sequences from D18S51, DYS389 I/II, and the second half of HV1 were successfully partitioned and analyzed. The ability to routinely deconvolute mtDNA mixtures down to a level of 1:250 allows for high resolution analysis of mtDNA heteroplasmy, and for differentiation of individuals from the same maternal lineage. The pyrosequencing approach results in poor resolution of homopolymeric sequences, and PCR/sequencing artifacts require a filtering mechanism similar to that for STR stutter and spectral bleed through. In addition, chimeric sequences from jumping PCR must be addressed to make the method operational.
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影响因子：
4.4
作者：
Fendt L;Zimmermann B;Daniaux M;Parson W
通讯作者：
Parson W
影响因子：
64.8
作者：
HIGUCHI, R;VONBEROLDINGEN, CH;ERLICH, HA
通讯作者：
ERLICH, HA
影响因子：
2.2
作者：
Budowle, B;Wilson, MR;Monson, KL
通讯作者：
Monson, KL
影响因子：
9.8
作者：
Li, Mingkun;Schoenberg, Anna;Stoneking, Mark
通讯作者：
Stoneking, Mark
DOI：
10.1007/978-1-59745-521-3_21
发表时间：
2009-01-01
期刊：
MITOCHONDRIAL DNA: METHODS AND PROTOCOLS
影响因子：
--
作者：
Kraytsberg, Yevgenya;Bodyak, Natalya;Khrapko, Konstantin
通讯作者：
Khrapko, Konstantin