Tailed pooled suppression subtractive hybridization (PSSH) adaptors do not alter efficiency.
Tailed pooled suppression subtractive hybridization (PSSH) adaptors do not alter efficiency.
复制标题
加尾合并抑制消减杂交 (PSSH) 接头不会改变效率。
DOI：
10.1007/s10482-010-9465-x
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Gill,StevenR
中科院分区：
文献类型：
--
作者：
Gerrish,RobertS;Gill,StevenR
抑制性消减杂交 (SSH) 及其衍生物合并抑制性消减杂交 (PSSH) 是用于研究原核基因组结构中大于 ~100 bp 变异的强大工具。最初的步骤包括将已知序列的寡核苷酸（“适配器”）连接到片段化的基因组上，以促进扩增、消减和下游测序。 SSH 创建了一个独特的 DNA 片段库，传统上通过桑格测序对其进行分析。许多下一代测序技术已进入市场，但 SSH 与这些平台不兼容。这是由于用于 SSH 的寡核苷酸具有高度的序列保守性。这种严格的粘附部分是因为尚未确定该寡核苷酸的改变是否会对消减效率产生有害影响。当非独特片段被需要精确核苷酸序列的二级自配对结构抑制时，就会发生扣除。我们确定在 PSSH 的嵌套 PCR 阶段将自定义序列附加到这些寡核苷酸的 5' 末端是否会降低扣除效率。我们比较了十个池子。使用标准 PSSH 和定制加尾 PSSH 进行金黄色葡萄球菌临床分离。我们发现它们的减法效率之间没有统计学上的显着差异。我们的观察表明，接头的末端可能在嵌套 PCR 步骤中被标记。这会生成标有自定义序列的库。这不会导致减法效率损失，并且对于希望将 SSH 或 PSSH 与自己的下游应用程序（例如高通量测序平台）结合起来的团体来说非常宝贵。
Suppression Subtractive Hybridization (SSH) and its derivative, Pooled Suppression Subtractive hybridization (PSSH), are powerful tools used to study variances larger than ~100 bp in prokaryotic genome structure. The initial steps involve ligating an oligonucleotide of known sequence (the “adaptor”) to a fragmented genome to facilitate amplification, subtraction and downstream sequencing. SSH results in the creation of a library of unique DNA fragments which have been traditionally analyzed via Sanger sequencing. Numerous next generation sequencing technologies have entered the market yet SSH is incompatible with these platforms. This is due to the high level of sequence conservation of the oligonucleotide used for SSH. This rigid adherence is partly because it has yet to be determined if alteration of this oligonucleotide will have a deleterious impact on subtraction efficiency. The subtraction occurs when non-unique fragments are inhibited by a secondary self-pairing structure which requires exact nucleotide sequence. We determine if appending custom sequence to the 5′ terminal ends of these oligonucleotides during the nested PCR stages of PSSH will reduce subtraction efficiency. We compare a pool of tenS. aureusclinical isolates with a standard PSSH and custom tailed-PSSH. We detected no statistically significant difference between their subtraction efficiencies. Our observations suggest that the adaptor’s terminal ends may be labeled during the nested PCR step. This produces libraries labeled with custom sequence. This does not lead to loss of subtraction efficiency and would be invaluable for groups wishing to combine SSH or PSSH with their own downstream applications, such as a high throughput sequencing platform.
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DOI：
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