. 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 L. 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.
This proposal deals with two issues in bioanalysis. We will first develop a novel and powerful tool for characterization of the microbiome. The technology developed in this grant will have obvious application in diseases of the gut. The second technology under development is to improve high sensitivity proteomic analysis, which will have applications whenever minute samples are to be analyzed, such as fine needle biopsies.
