适用于下一代测序的植物细菌DNA富集方法的优化与比较分析
Summary
提出了两种植物细菌DNA富集方法的比较和优化：传统差速离心和基于甲基化状态的总gDNA分馏。我们评估产生的DNA数量和质量，证明在短读下一代测序中的性能，并讨论长读单分子测序中使用的潜力。
Abstract
植物细胞器基因组包含大的重复元件，其可能经历配对或重组以形成复合结构和/或亚基因组片段。细胞器基因组也存在于给定细胞或组织类型（异质性）内的混合物中，并且丰富的亚型可能在整个发育过程中或当处于应力（亚化学计量转移）时）发生变化。需要下一代测序（NGS）技术来更深入地了解细胞器基因组结构和功能。传统的测序研究使用几种方法来获得细胞器DNA：（1）如果使用大量的起始组织，将其均质化并进行差速离心和/或梯度纯化。 （2）如果使用较少量的组织（ 即如果种子，材料或空间有限），则按照（1）所述进行相同的处理，然后进行全基因组扩增以获得足够的DNA。 （3）生物信息学分析可用于分析使用总基因组DNA并解析出细胞器读数。所有这些方法都有固有的挑战和权衡。在（1）中，可能难以获得如此大量的起始组织;在（2）中，全基因组扩增可能引入测序偏倚;在（3）中，核和细胞器基因组之间的同源性可能会干扰组装和分析。在具有大核基因组的植物中，富集细菌DNA以降低生物信息学分析的测序成本和序列复杂度是有利的。在这里，我们将传统的差速离心法与第四种方法（适应性CpG-甲基下拉法）进行比较，将总基因组DNA分离成核和细胞器级分。两种方法为NGS产生足够的DNA，对于细胞器序列高度富集的DNA，尽管在线粒体和叶绿体中具有不同的比例。我们介绍了这些方法对小麦叶组织的优化，并讨论了主要优点和d在样本输入，协议易用性和下游应用的上下文中的每种方法的优点。
Introduction
基因组测序是解析重要植物性状的潜在遗传基础的有力工具。大多数基因组测序研究集中于核基因组内容，因为大多数基因位于细胞核中。然而，细胞器基因组，包括线粒体（跨真核生物）和质（植物;在特殊形式，叶绿体，工作在光合作用）促进生物发育，应激反应和整体健康至关重要的1显著的遗传信息。细胞器基因组通常包括在用于核基因组测序的总DNA的提取，虽然方法，以减少前，DNA提取的细胞器号码也采用2。许多研究已经使用测序结果从总的gDNA提取组装细胞器基因组3，4，5，外部参照“> 6,7。然而，当研究的目标是把重点放在细胞器基因组，使用总的gDNA增加了测序成本，因为很多的读取，但‘丢失’的核的DNA序列，特别是在植物具有大核基因组此外，由于重复和传输细胞器序列的进核基因组和细胞器之间，解决测序的正确的映射位置读取到适当的基因组是生物信息学有挑战性2,8。细胞器基因组的从核基因组的纯化是一个减少这些问题的策略，进一步的生物信息学策略可用于分离映射到线粒体和叶绿体之间同源区域的读数。
虽然来自许多植物物种的细胞器基因组已被测序，但是对于细胞器基因组多样性的广度几乎不了解可用于野生种群或栽培育种池。还知道细胞器基因组是由于重复序列9之间的重组而经历显着的结构重排的动态分子。此外，每个细胞器中含有多个拷贝的细胞器基因组，并且每个细胞内都含有多个细胞器。并非所有这些基因组的拷贝都是相同的，这被称为异质性。与“主圈”的规范图相比，现在越来越多的证据表明，细胞基因组结构更复杂，包括亚基因组圆，线性染色体，线性连接子和支链结构10 。植物细胞器基因组的组装由于其相对较大的尺寸和大量的反向和直接重复而进一步复杂化。
用于细胞器分离，DNA纯化和随后基因组的传统方案 È测序往往笨重且需要大体积的组织输入的，具有几克到几百个向上必要为起点11，12，13，14，15，16，17克幼叶组织的组成。当组织受限时，这使得细胞器基因组测序不可及。在某些情况下，种子数量有限，例如当需要在代际基础上进行序列或在必须通过杂交维持的雄性不育系中时。在这些情况下，可以纯化细胞器DNA，然后进行全基因组扩增。然而，全基因组扩增可以引入显着的测序偏差，这在评估结构变异，亚基因组结构和异质性水平时是一个特别的问题> 18。用于短读序列技术的图书馆准备工作的最新进展克服了低输入障碍，以避免全基因组扩增。例如，Illumina Nextera XT库制备试剂盒允许使用少至1ng的DNA作为输入19 。然而，用于长读取测序应用（如PacBio或Oxford Nanopore测序技术）的标准库制备仍然需要相对较高量的输入DNA，这可能对组织基因组测序构成挑战。最近，新的用户制作，长读测序的协议已被开发，以减少输入量和帮助促进在获得的DNA微克，批量样品的基因组测序是困难的20，21。然而，获得高分子量纯的细胞器级分以进入这些文库制剂仍是一个挑战。
我们寻求o比较和优化不需要全基因组扩增的适用于NGS的细胞器DNA富集和分离方法。具体来说，我们的目标是确定从有限起始材料（如叶子子样品）中丰富高分子量细胞器DNA的最佳实践。这项工作提供了丰富细胞DNA的方法的比较分析：（1）修改的传统差速离心方案与（2）基于使用市售DNA CpG-甲基结合结构域蛋白质下拉法的DNA分级方案22施用于植物组织23 。我们建议从小麦叶组织中分离细胞叶DNA的最佳实践，这可能容易地扩展到其他植物和组织类型。
Protocol
1.生成用于细胞分离和DNA提取的植物材料 小麦幼苗的标准生长 在每个角落有4-6个种子的小方形花盆中种植蛭石。转移到温室或生长室，16小时光周期，23ºC/ 18ºC夜间。 每天浇灌植物。在萌发和发芽后7天，用¼茶匙颗粒状20-20-20 NPK肥料施肥。 小麦幼苗的替代品种 按照步骤1.1，但将盆置于暗生长室中，23℃16小时/ 18℃8小时。或者，?…
Representative Results
本手册中提出的方案描述了从植物组织中丰富细胞器DNA的两种不同的方法。这里提出的条件反映了小麦组织的优化。 图1中描述了协议，组织输入和DNA输出中的关键步骤的比较。我们测试的DC协议的步骤遵循与先前描述的相似的条件（ 图1A ）。收获的组织必须新鲜处理，并进行差速离心和/或梯度以分离完整的细胞器。在细胞器?…
Discussion
迄今为止，大多数细胞器测序研究集中于传统的DC方法，以丰富特定的DNA。已经描述了从不同植物中分离细胞器的方法，包括苔藓40 ;单子叶植物如小麦15和燕麦11 ;和双子叶植物如拟南芥11 ，向日葵17和油菜籽14 。大多数协议集中在叶组织<s…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要承认美国农业部农业研究服务处和国家科学基金会（IOS 1025881和IOS 1361554）的资助。感谢R. Caspers进行温室维护和植物保养。我们还感谢明尼苏达大学基因组学中心，其中进行了Illumina图书馆的制备和测序。我们也感谢期刊编辑和四位匿名审稿人的意见，进一步加强了我们的稿件。我们也感谢经合组织与SK的合作，将这些协议项目与日本的同事整合起来。
Materials
| 2-mercaptoethanol (beta-mercaptoethanol; BME) | Sigma Aldrich | M3148-100ml | |
| 2-propanol (Isopropyl alcohol/isopropanol), bioreagent | Sigma Aldrich | I9516 | |
| agarose, Bio-Rad Cetified Megabase agarose | Bio-Rad | 1613108 | |
| analytical balance | Mettler Toledo | AB54-S | |
| balance | Mettler Toledo | PB1502-S | |
| bovine serum albumin (BSA) | Sigma Aldrich | B4287-25G | |
| Ceramic grinding cylinders, 3/8in x 7/8in | SPEX SamplePrep | 2183 | |
| Cryogenic Blocks compatible with tissue homogenizer for holding 50 ml tubes | SPEX SamplePrep | 2664 | |
| DNaseI | Sigma | DN25 | |
| ethanol, absolute | Decon Laboratories | 2716 | |
| Ethylenediamine Tetraacetic Acid (EDTA), 0.5M Solution, pH8.0 | Fisher | BP2482-500 | |
| gel imaging system | |||
| gel stain | Such as GelRed or Ethidium Bromide | ||
| grinding pestle, wide tip for 2 ml conical tubes | |||
| Guanidine-HCl, 8M solution | ThermoFisher | 24115 | |
| LightCycler 480 SYBR Green I Master | Roche | 4707516001 | |
| liquid nitrogen | |||
| Lysing enzymes from Trichoderma harzianum | Sigma | L1412 | |
| Magnesium Chloride | G Bioscience | 24115 | |
| magnetic rack | ThermoFisher | A13346 | |
| microcentrifuge tubes, LoBind 1.5 ml | Eppendorf | 22431021 | |
| microcentrifuge tubes, standard nuclease-free 1.5 ml | Eppendorf | ||
| microcentrifuge, refrigerated | Sorvall | Legend X1R | Or equivalent product, must be capable of reaching at least 18,000 x g with rotors for 50 ml tubes, Oak Ridge tubes, and 1.5 ml tubes |
| microcentrifuge, room temperature | Eppendorf | 5424 | Or equivalent product, must be capable of reaching at least 18,000 x g with rotor for 1.5 ml and 2 ml microcentrifuge tubes |
| Microcon DNA Fast Flow Centrifugal Filter Units | EMD Millipore | MRCFOR100 | |
| Miracloth, 1 square per sample cut to fit funnel | EMD Millipore | 475855 | |
| NEBNext Microbiome DNA Enrichment Kit | New England Biolabs | E2612L | |
| parafilm | Parafilm M | PM992 | |
| plastic pots and trays | |||
| polyvinylpyrrolidone (PVP) | Fisher | BP431-100 | |
| Proteinase K | Qiagen | 19131 | |
| Pulsed-Field Gel Electrophoresis rig (e.g. CHEF DR III) | Bio-Rad | 1703697 | |
| purification beads, Agencourt AMpureXP beads | Beckman Coulter | A63881 | |
| QIAamp DNA Mini Kit | Qiagen | 51304 | |
| Qiagen 20/g Genomic Tip DNA Extraction Kit | Qiagen | 10223 | |
| Qiagen Buffer EB (elution buffer) | Qiagen | 19086 | |
| Qiagen DNA Extraction Buffer Set | Qiagen | 19060 | |
| QiaRack | Qiagen | 19015 | |
| qPCR machine (e.g. Roche Light Cycler 480) | Roche | ||
| qPCR plate sealing film | Roche | 4729757001 | |
| qPCR plate, 96 well plate | Roche | 4729692001 | |
| Qubit assay tubes | Life Technologies | Q32856 | |
| Qubit Broad Spectrum assay kit | Life Technologies | Q32850 | |
| Qubit High Sensitivity assay kit | Life Technologies | Q32851 | |
| RNaseA | Qiagen | 19101 | |
| Serological pipettes (20 ml) and pipet-aid | Fisher | 13-678-11 | |
| Small funnels, 1 per sample | |||
| Sodium Chloride | Ambion | AM9759 | |
| Soft paintbrush, 2 per sample | |||
| SPEX SamplePrep 2010 Geno/Grinder or another type of tissue homogenizer | SPEX SamplePrep | Or another comparable tissue homogenizer. If you do not have access to a tissue homogenizer, then grinding in a pre-chilled mortar and pestle will suffice (see protocol for details). However, a homogenizer will give more consistent results and total homogenization time is reduced. | |
| Sucrose | Omnipure | 8550 | |
| TBE | |||
| thermomixer | |||
| Tris | Sigma | T2819-100ml | |
| Triton X-100 | Promega | H5142 | |
| tube rotater | |||
| tubes, 50 mL conical polypropylene | Corning | 352070 | |
| tubes, 50 ml high-speed polypropylene | ThermoScientific/Nalgene | 3119-0050 | e.g. Nalgene Oakridge tubes or equivalent |
| vermiculite | |||
| water bath | |||
| water, sterile and certified Nuclease-free | Fisher | 1481 | |
| water, sterile milliQ |
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Citar este artigo
Miller, M. E., Liberatore, K. L., Kianian, S. F. Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing. J. Vis. Exp. (125), e55528, doi:10.3791/55528 (2017).