Advances in bioanalysis

抽象的。这MIRA提案是R 01 GM 096767的延续，该提案最近开始了第八次 年融资。已经发表了64篇论文，感谢这项赠款的支持。 拟议的工作有两大主题： 第一个主题解决了微生物组宏基因组分析中固有的低效率。 宏基因组学是一种不依赖培养的微生物群落研究技术。在metag- 在基因组学研究中，微生物组中所有生物的基因组都被提取、剪切， 艾德进入下一代测序。然后将这些序列组装成重叠群并进行映射 到基因组目前的技术是非常低效的，因为基因组来自丰富的 物种不可避免地主导测序数据。因此，需要庞大的数据集来解决 低丰度类群的基因组。此外，来自相关物种和菌株的序列混淆了 准确的装配。为了解决这些问题，我们使用毛细管区带电泳（CZE）来分离- 将复杂废水微生物组的等分试样吃入微量滴定板的威尔斯孔中， IC分析分级分离在微量滴定板的几个威尔斯孔中高度丰富的种类， 允许在其它威尔斯孔中成功测序稀有物种。我们已经证明，CZE 分级分离可以使分辨的分类群的数量增加5.8倍， 在属的水平上解决的分类群的数量，在属的水平上解决的分类群的数量增加了30倍， 在物种水平上，每个属的基因数量增加了13倍，而每个属的基因数量增加了23倍。 与普通微生物组的常规分析相比，每个物种的基因数量。 我们证明，CZE解决细菌菌株成不同的馏分。我们提出了一个系统的 研究分离模式和条件，以进一步增加已鉴定的属、种、 cies和strains。 第二个主题是高灵敏度和高通量蛋白质分析。我们已经开发 基于微升体积微反应器的样品制备方法 样品制备的转移步骤;清洁和洗脱样品所需的试剂体积， 两者均<3 μL。微反应器的小尺寸、低试剂体积和少量的SAM- 简单的处理步骤大大提高了亚微克样品的回收率。我们使用UPLC-ESI 与Q-Exactive HF质谱仪耦合，用于分析处理后的样品;该系统具有 从单个非洲爪蟾50期中鉴定了20，943个独特的肽和2，597个蛋白质组 卵裂球我们的目标是研究替代的分离方法，并将这项技术扩展到 192个非洲爪蟾胚胎卵裂球的平行处理。

Abstract. This MIRA proposal is a continuation of R01GM096767, which recently began its eighth year of funding. 64 papers have been published that acknowledge support from this grant. Two overarching themes form the proposed work: The first theme addresses the inherent inefficiencies in metagenomic analysis of microbiomes. Metagenomics is a culture-independent technique for the study of microbial communities. In metag- enomic studies, the genomes of all organisms in a microbiome are extracted, sheared, and subject- ed to next-generation sequencing. The sequences are then assembled into contigs and mapped to genomes. Current technology is extraordinarily inefficient because the genomes from abundant species inevitably dominate the sequencing data. As a result, huge data sets are needed to resolve genomes of low-abundance taxa. In addition, sequences from related species and strains confound accurate assembly. To address these issues, we use capillary zone electrophoresis (CZE) to fraction- ate an aliquot of a complex wastewater microbiome into wells of a microtiter plate before metagenom- ic analysis. Fractionation segregates highly abundant species in a few wells of the microtiter plate, allowing successful sequencing of rarer species in other wells. We have demonstrated that CZE fractionation can produce a 5.8-fold increase in the number of resolved taxa, a three-fold increase in the number of taxa resolved at the genus level, a 30-fold increase in the number of taxa resolved at the species level, a 13-fold increase in the number of genes per genus, and a 23-fold increase in the number of genes per species compared with conventional analysis of the unfractionated microbiome. We demonstrate that CZE resolved bacterial strains into different fractions. We propose a systematic study of separation modes and conditions to further increase the numbers of identified genus, spe- cies, and strains. The second theme is high sensitivity and high-throughput protein analysis. We have developed a method for sample preparation based on a microliter volume microreactor that uses two sample transfer steps for sample preparation; the reagent volumes required for cleaning and eluting the sam- ple are both <3 µL. The small size of the microreactor, low reagent volume, and small number of sam- ple processing steps greatly improve the recovery of sub-microgram samples. We use a UPLC-ESI coupled with a Q-Exactive HF mass spectrometer for analysis of processed sample; the system has identified 20,943 unique peptides and 2,597 protein groups from a single Xenopus laevis stage 50 blastomere. Our goal is to investigate alternative separation methods and to extend this technology to the parallel processing of 192 blastomeres taken from Xenopus laevis embryos.