Advances in bioanalysis

抽象的。这个Mira提案是R01GM096767的延续，R01GM096767最近开始了第八次 一年的资金。的论文已经发表，承认这笔资金的支持。 两个主要主题构成了拟议的工作： 第一个主题涉及微生物群元基因组分析中固有的低效问题。 元基因组学是一种独立于培养物的微生物群落研究技术。在元标记中- 在经济研究中，微生物组中所有生物的基因组被提取、剪切并研究- ED到下一代测序。然后将这些序列组装成重叠群并进行映射 到基因组。目前的技术效率非常低，因为来自丰富的基因组 物种不可避免地在测序数据中占据主导地位。因此，需要解决庞大的数据集 低丰度分类群的基因组。此外，来自相关物种和菌株的序列也混淆了 装配精准。为了解决这些问题，我们使用毛细管区带电泳(CZE)来分离- 在元基因组之前，将一份等量的复杂废水微生物群放入微滴定板的孔中- IC分析。分馏在微量滴定板的几个孔中分离出高度丰富的物种， 从而成功地对其他油井中的稀有物种进行了测序。我们已经证明了CZE 分馏可以使已分解的分类群数量增加5.8倍，增加3倍 在属水平上分解的分类群数量，在属水平上分解的分类群数量增加30倍 物种水平，每个属的基因数量增加了13倍， 每个物种的基因数量与常规的未分离微生物组分析相比较。 我们证明了CZE将细菌菌株分解成不同的组分。我们提出了一种系统的 进一步增加已鉴定属的分离方式和条件的研究。 CIES和SRANSE。 第二个主题是高灵敏度和高通量的蛋白质分析。我们已经开发出 一种基于两个样品的微升体积微反应器的样品制备方法 样品制备的转移步骤；清洗和洗脱样品所需的试剂体积- 微反装置体积小，试剂用量少，样品量少。 PLE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质谱仪相结合，用于分析处理后的样品；该系统具有 从一只非洲爪哇中鉴定出20,943个独特的多肽和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 非洲爪哇胚胎192个卵裂球的平行处理

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.