High-Resolution Differentiation of Enteric Bacteria in Premature Infant Fecal Microbiomes Using a Novel rRNA Amplicon.

Identifying and tracking microbial strains as microbiomes evolve are major challenges in the field of microbiome research. We utilized a new sequencing kit that combines DNA extraction with PCR amplification of a large region of the rRNA operon and downstream bioinformatic data analysis. Longitudinal microbiome samples of coadmitted twins from two different neonatal intensive care units (NICUs) were analyzed using an ∼2,500-base amplicon that spans the 16S and 23S rRNA genes and mapped to a new, custom 16S-23S rRNA database. Amplicon sequence variants (ASVs) inferred using DADA2 provided sufficient resolution for the differentiation of rRNA variants from closely related but not previously sequenced Klebsiella, Escherichia coli, and Enterobacter strains, among the first bacteria colonizing the gut of these infants after admission to the NICU. Distinct ASV groups (fingerprints) were monitored between coadmitted twins over time, demonstrating the potential to track the source and spread of both commensals and pathogens. The high-resolution taxonomy obtained from long amplicon sequencing enables the tracking of strains temporally and spatially as microbiomes are established in infants in the hospital environment.IMPORTANCE Achieving strain-level resolution is a major obstacle for source tracking and temporal studies of microbiomes. In this study, we describe a novel deep-sequencing approach that provides species- and strain-level resolution of the neonatal microbiome. Using Klebsiella, E. coli, and Enterobacter as examples, we could monitor their temporal dynamics after antibiotic treatment and in pairs of twins. The strain-level resolution, combined with the greater sequencing depth and decreased cost per read of PacBio Sequel 2, enables this advantageous source- and strain-tracking analysis method to be implemented widely across more complex microbiomes.

Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii.

Escherichia coli, Escherichia albertii, and Escherichia fergusonii are closely related bacteria that can cause illness in humans, such as bacteremia, urinary tract infections and diarrhea. Current identification strategies for these three species vary in complexity and typically rely on the use of multiple phenotypic and genetic tests. To facilitate their rapid identification, we developed a multiplex PCR assay targeting conserved, species-specific genes. We used the Daydreamer™ (Pattern Genomics, USA) software platform to concurrently analyze whole genome sequence assemblies (WGS) from 150 Enterobacteriaceae genomes (107 E. coli, 5 Shigella spp., 21 E. albertii, 12 E. fergusonii and 5 other species) and design primers for the following species-specific regions: a 212bp region of the cyclic di-GMP regulator gene (cdgR, AW869_22935 from genome K-12 MG1655, CP014225) for E. coli/Shigella; a 393bp region of the DNA-binding transcriptional activator of cysteine biosynthesis gene (EAKF1_ch4033 from genome KF1, CP007025) for E. albertii; and a 575bp region of the palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase (EFER_0790 from genome ATCC 35469, CU928158) for E. fergusonii. We incorporated the species-specific primers into a conventional multiplex PCR assay and assessed its performance with a collection of 97 Enterobacteriaceae strains. The assay was 100% sensitive and specific for detecting the expected species and offers a quick and accurate strategy for identifying E. coli, E. albertii, and E. fergusonii in either a single reaction or by in silico PCR with sequence assemblies.

A fast boosting-based screening method for large-scale association study in complex traits with genetic heterogeneity.

Genome-wide association study for complex diseases will generate massive amount of single nucleotide polymorphisms (SNPs) data. Univariate statistical test (i.e. Fisher exact test) was used to single out non-associated SNPs. However, the disease-susceptible SNPs may have little marginal effects in population and are unlikely to retain after the univariate tests. Also, model-based methods are impractical for large-scale dataset. Moreover, genetic heterogeneity makes the traditional methods harder to identify the genetic causes of diseases. A more recent random forest method provides a more robust method for screening the SNPs in thousands scale. However, for more large-scale data, i.e., Affymetrix Human Mapping 100K GeneChip data, a faster screening method is required to screening SNPs in whole-genome large scale association analysis with genetic heterogeneity. We propose a boosting-based method for rapid screening in large-scale analysis of complex traits in the presence of genetic heterogeneity. It provides a relatively fast and fairly good tool for screening and limiting the candidate SNPs for further more complex computational modeling task.

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

A whole-genome assembly of Drosophila.

We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.

An algorithmic approach to multiple complete digest mapping.

Multiple Complete Digest (MCD) mapping is a method of determining the locations of restriction sites along a target DNA molecule. The resulting restriction map has many potential applications in DNA sequencing and genetics. In this work, we present a heuristic algorithm for fragment identification, a key step in the process of constructing an MCD map. Given measurements of the restriction fragment sizes from one or more complete digestions of each clone in a clone library covering the molecule to be mapped, the algorithm identifies groups of restriction fragments on different clones that correspond to the same region of the target DNA. Once these groups are correctly determined the desired map can be constructed by solving a system of simple linear inequalities. We demonstrate the effectiveness of our algorithm on real data provided by the Genome Center at the University of Washington.

Effects of oral and parenteral selenium supplements on residues in meat, milk and eggs.

Oral and parenteral preparations of Se are used worldwide to prevent and treat nutritional muscular dystrophy and other Se deficiency syndromes. There are extensive published data on the effects of oral supplementation on Se residues in food animal products. Very little published data exist on the effects of parenteral administration on Se residues, even for cattle and swine in which parenteral preparations are used extensively. The distribution of Se into kidney and liver appears to be equivalent for both forms of supplementation. Elimination of Se in milk is greater after parenteral administration and correlates with high plasma Se levels, however the milk excretion drops quickly and after 4 d returns to control levels (Little et al. 1979). Of particular interest is the finding that up to 18% of Se in an oral diet may be excreted in milk (Maus et al. 1980). Use of Se supplements in poultry results in increased levels of Se in liver, kidney, and eggs. Distribution of Se into liver and kidney is much greater than into breast muscle indicating a greater capacity of these organs to accumulate Se. Excretion of Se into eggs results in Se levels equivalent to those in liver and kidney, indicating that eggs are an important route of Se excretion in laying hens (Ort and Latshaw 1978). When Se supplementation stops, the liver, kidney, and egg white and yolk residues decline quickly to control values within 1-2 wk. Breast muscle Se content changes little during supplementation and after withdrawal of supplementation. Oral and parenteral selenium supplementation in swine result in greater accumulation of Se in liver and kidney than in muscle. Oral selenium supplementation also increases the excretion of Se into milk. This method has been used to prevent Se deficiency disease in piglets (Mahan et al. 1975). Oral supplementation with 0.1 ppm Se, as sodium selenate, did not result in levels of Se in blood, meat, or viscera at slaughter (Jenkins and Winter 1973). Despite the large amount of data available on Se residues in food animals, additional information on the pharmacokinetics of parenterally administered Se preparations is needed, especially in sheep and goats which receive parenteral Se supplements. Information on the disappearance kinetics of Se residues in meat and milk is needed for all ruminants. The data currently available in the literature does not allow the calculation of pharmacokinetic parameters of Se in any species. Properly performed pharmacokinetic studies would contribute a great deal towards a better understanding of how food animals utilize supplemental selenium.(ABSTRACT TRUNCATED AT 400 WORDS)