Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences

The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to capture more than 90% of sequences in the Greengenes database and with nearly twice the resolution of existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the diversity of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.Comment: 17 pages, 2 tables, 2 figures, supplementary materia

Isolation of 39 polymorphic microsatellite loci and the development of a fluorescently labelled marker set for the Eurasian badger

We have isolated 78 microsatellite loci from the Eurasian badger (Meles meles). Of the 52 loci characterized, 39 were found to be polymorphic. A fluorescently labelled primer set was developed to enable individual-specific 17-locus genotypes to be obtained efficiently

Blue cone monochromacy: causative mutations and associated phenotypes.

PurposeTo perform a phenotypic assessment of members of three British families with blue cone monochromatism (BCM), and to determine the underlying molecular genetic basis of disease.MethodsAffected members of three British families with BCM were examined clinically and underwent detailed electrophysiological and psychophysical testing. Blood samples were taken for DNA extraction. Molecular analysis involved the amplification of the coding regions of the long (L) and medium (M) wave cone opsin genes and the upstream locus control region (LCR) by polymerase chain reaction (PCR). Gene products were directly sequenced and analyzed.ResultsIn all three families, genetic analysis identified that the underlying cause of BCM involved an unequal crossover within the opsin gene array, with an inactivating mutation. Family 1 had a single 5'-L-M-3' hybrid gene, with an inactivating Cys203Arg (C203R) mutation. Family 3 had an array composed of a C203R inactivated 5'-L-M-3' hybrid gene followed by a second inactive gene. Families 1 and 3 had typical clinical, electrophysiological, and psychophysical findings consistent with stationary BCM. A novel mutation was detected in Family 2 that had a single hybrid gene lacking exon 2. This family presented clinical and psychophysical evidence of a slowly progressive phenotype.ConclusionsTwo of the BCM-causing family genotypes identified in this study comprised different hybrid genes, each of which contained the commonly described C203R inactivating mutation. The genotype in the family with evidence of a slowly progressive phenotype represents a novel BCM mutation. The deleted exon 2 in this family is not predicted to result in a shift in the reading frame, therefore we hypothesize that an abnormal opsin protein product may accumulate and lead to cone cell loss over time. This is the first report of slow progression associated with this class of mutation in the L or M opsin genes in BCM

Matrilines in Neolithic cattle from Orkney, Scotland reveals complex husbandry patterns of ancestry

mtDNA, isotopic and archaeozoological analyses of cattle teeth and bones from the Late Neolithic site of Links of Noltland, Orkney, Scotland revealed these animals followed similar grazing regimes but displayed diverse genetic origins and included one cattle skull that carried an aurochs (wild cattle) genetic haplotype. Morphometric analyses indicate the presence of some cattle larger than published dimensions of Neolithic domestic cattle. Several explanations for these finding are possible but may be the evidence of a complex pattern of domestic cattle introductions into Neolithic Orkney and interbreeding between domestic and wild cattle

Differentiable structures on metric measure spaces: A Primer

This is an exposition of the theory of differentiable structures on metric measures spaces, in the sense of Cheeger and Keith.Comment: 23 page

The Rapid and Sensitive Quantitative Determination of Galactose by Combined Enzymatic and Colorimetric Method: Application in Neonatal Screening

The quantitative measurement of galactose in blood is essential for the early diagnosis, treatment, and dietary monitoring of galactosemia patients. In this communication, we aimed to develop a rapid, sensitive, and cost-effective combined method for galactose determination in dry blood spots. This procedure was based on the combination of enzymatic reactions of galactose dehydrogenase (GalDH), dihydrolipoyl dehydrogenase (DLD), and alkaline phosphates with a colorimetric system. The incubation time and the concentration of enzymes used in new method were also optimized. The analytical performance was studied by the precision, recovery, linearity, and sensitivity parameters. Statistical analysis was applied to method comparison experiment. The regression equation and correlation coefficient (R2) were Y = 0.0085x + 0.032 and R2 = 0.998, respectively. This assay exhibited a recovery in the range of 91.7–114.3 % and had the limit detection of 0.5 mg/dl for galactose. The between-run coefficient of variation (CV) was between 2.6 and 11.1 . The within-run CV was between 4.9 and 9.2 . Our results indicated that the new and reference methods were in agreement because no significant biases exist between them. Briefly, a quick and reliable combined enzymatic and colorimetric assay was presented for application in newborn mass screening and monitoring of galactosemia patients. © 2016 Springer Science+Business Media New Yor

Gastrointestinal tract size, total-tract digestibility, and rumen microflora in different dairy cow genotypes

peer-reviewedThe superior milk production efficiency of Jersey (JE) and Jersey × Holstein-Friesian (JE × HF) cows compared with Holstein-Friesian (HF) has been widely published. The biological differences among dairy cow genotypes, which could contribute to the milk production efficiency differences, have not been as widely studied however. A series of component studies were conducted using cows sourced from a longer-term genotype comparison study (JE, JE × HF, and HF). The objectives were to (1) determine if differences exist among genotypes regarding gastrointestinal tract (GIT) weight, (2) assess and quantify whether the genotypes tested differ in their ability to digest perennial ryegrass, and (3) examine the relative abundance of specific rumen microbial populations potentially relating to feed digestibility. Over 3 yr, the GIT weight was obtained from 33 HF, 35 JE, and 27 JE × HF nonlactating cows postslaughter. During the dry period the cows were offered a perennial ryegrass silage diet at maintenance level. The unadjusted GIT weight was heavier for the HF than for JE and JE × HF. When expressed as a proportion of body weight (BW), JE and JE × HF had a heavier GIT weight than HF. In vivo digestibility was evaluated on 16 each of JE, JE × HF, and HF lactating dairy cows. Cows were individually stalled, allowing for the total collection of feces and were offered freshly cut grass twice daily. During this time, daily milk yield, BW, and dry matter intake (DMI) were greater for HF and JE × HF than for JE; milk fat and protein concentration ranked oppositely. Daily milk solids yield did not differ among the 3 genotypes. Intake capacity, expressed as DMI per BW, tended to be different among treatments, with JE having the greatest DMI per BW, HF the lowest, and JE × HF being intermediate. Production efficiency, expressed as milk solids per DMI, was higher for JE than HF and JE × HF. Digestive efficiency, expressed as digestibility of dry matter, organic matter, N, neutral detergent fiber, and acid detergent fiber, was higher for JE than HF. In grazing cows (n = 15 per genotype) samples of rumen fluid, collected using a transesophageal sampling device, were analyzed to determine the relative abundance of rumen microbial populations of cellulolytic bacteria, protozoa, and fungi. These are critically important for fermentation of feed into short-chain fatty acids. A decrease was observed in the relative abundance of Ruminococcus flavefaciens in the JE rumen compared with HF and JE × HF. We can deduce from this study that the JE genotype has greater digestibility and a different rumen microbial population than HF. Jersey and JE × HF cows had a proportionally greater GIT weight than HF. These differences are likely to contribute to the production efficiency differences among genotypes previously reported

A primer on noise-induced transitions in applied dynamical systems

Noise plays a fundamental role in a wide variety of physical and biological dynamical systems. It can arise from an external forcing or due to random dynamics internal to the system. It is well established that even weak noise can result in large behavioral changes such as transitions between or escapes from quasi-stable states. These transitions can correspond to critical events such as failures or extinctions that make them essential phenomena to understand and quantify, despite the fact that their occurrence is rare. This article will provide an overview of the theory underlying the dynamics of rare events for stochastic models along with some example applications

Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types

Culture-adapted Plasmodium falciparum isolates from UK travellers: in vitro drug sensitivity, clonality and drug resistance markers.

BACKGROUND: The screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy. METHODS: Pre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC₅₀ estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines. RESULTS: Plasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC₅₀ estimates, and carried the CVIET haplotype at codons 72-76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC₅₀ estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86. CONCLUSIONS: This study describes the establishment in continuous culture, in vitro drug sensitivity testing and molecular characterization of a series of multiclonal P. falciparum isolates taken directly from UK malaria patients following recent travel to various malaria-endemic countries in Africa. These "HL" isolates are available as an open resource for studies of drug response, antigenic diversity and other aspects of parasite biology