Analysis of the Rumen Microbiota of Beef Calves Supplemented During the Suckling Phase

A study was conducted to examine the effects of supplementing beef calves during their suckling phase (popularly known as creep feeding) with supplements that contained or did not contain the enzyme xylanase. Forty-two cow-calf pairs were divided into three groups and assigned to one of three treatments for a period of 105 days, as follows: (1) No supplemental feed for calves (control; CON); (2) Corn and soybean meal-based supplement feed for calves (positive control; PCON); and (3) Same feed regimen as PCON with xylanase added to the supplement (enzyme; ENZ). After 105 days, out of the 42 calves participating in the study, 25 male calves were randomly selected (8 from CON, 9 from PCON, and 8 from ENZ) and samples of their forestomach were collected by esophageal tubing. Immediately after this procedure, all calves were weaned, commingled, and placed in a common post-weaning diet for 4 weeks. At the end of this period, ruminal fluid was once again collected from the same 25 calves. All samples were subjected to DNA extraction and 16S rRNA gene sequencing. At weaning, most of the alpha diversity indexes were greater in CON; however, no differences (P ≥ 0.23) in alpha diversity were observed in samples collected 4 weeks after weaning. Regardless of treatment, 2 phyla – Bacteroidetes and Firmicutes – comprised approximately 80% of the total bacterial abundance of samples collected on both days. At the genus level, an effect of diet (P = 0.02) was observed for Prevotella in the samples collected at weaning; however, no differences were detected in the samples collected 4 weeks after weaning. Calf average daily gain (ADG) during the 105-day creep feeding trial tended (P = 0.09) to be greater in the groups that received supplementation, with the greatest numerical value observed in ENZ. Moreover, there was a positive correlation (ρ = 0.43; P = 0.03) between ADG and abundance of Prevotella, indicating the importance of this bacterial group for ruminants. In summary, most of the significant differences found in this study were detected at weaning, and the majority of them disappeared 4 weeks after the calves were weaned and commingled

Adapterama IV: Sequence Capture of Dual-digest RADseq Libraries with Identifiable Duplicates (RADcap)

AbstractMolecular ecologists seek to genotype hundreds to thousands of loci from hundreds to thousands of individuals at minimal cost per sample. Current methods such as restriction site associated DNA sequencing (RADseq) and sequence capture are constrained by costs associated with inefficient use of sequencing data and sample preparation, respectively. Here, we demonstrate RADcap, an approach that combines the major benefits of RADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches. The RADcap approach uses a new version of dual-digest RADseq (3RAD) to identify candidate SNP loci for capture bait design, and subsequently uses custom sequence capture baits to consistently enrich candidate SNP loci across many individuals. We combined this approach with a new library preparation method for identifying and removing PCR duplicates from 3RAD libraries, which allows researchers to process RADseq data using traditional pipelines, and we tested the RADcap method by genotyping sets of 96 to 384Wisteriaplants. Our results demonstrate that our RADcap method: 1) can methodologically reduce (to &lt;5%) and computationally remove PCR duplicate reads from data; (2) achieves 80-90% reads-on-target in 11 of 12 enrichments; (3) returns consistent coverage (≥4x) across &gt;90% of individuals at up to 99.9% of the targeted loci; (4) produces consistently high occupancy matrices of genotypes across hundreds of individuals; and (5) is inexpensive, with reagent and sequencing costs totaling &lt;$6/sample and adapter and primer costs of only a few hundred dollars.</jats:p

Genomic mutations after multigenerational exposure of Caenorhabditis elegans to pristine and sulfidized silver nanoparticles

Our previous study showed heritable reproductive toxicity in the nematode Caenorhabditis elegans after multigenerational exposure to AgNO3 and silver nanoparticles (Ag-NPs). The aim of this study was to determine whether such inheritable effects are correlated with induced germline mutations in C. elegans. Individual C. elegans lineages were exposed for 10 generations to equitoxic concentrations at EC30 of AgNO3, Ag-NPs, and sulfidized Ag-NPs (sAg-NPs), a predominant environmentally transformed product of pristine Ag-NPs. The mutations were detected via whole genome DNA sequencing approach by comparing F0 and F10 generations. An increase in the total number of variants, though not statistically significant, was observed for all Ag treatments and the variants were mainly contributed by single nucleotide polymorphisms (SNPs). This potentially contributed towards reproductive as well as growth toxicity shown previously after ten generations of exposure in every Ag treatment. However, despite Ag-NPs and AgNO3 inducing stronger reproductive toxicity than sAg-NPs, exposure to sAg-NPs resulted in higher mutation accumulation with significant increase in the number of transversions. Thus our results suggest that other mechanisms of inheritance, such as epigenetics, may be at play in Ag-NP- and AgNO3-induced multigenerational and transgenerational reproductive toxicity

Conflicting Evolutionary Histories of the Mitochondrial and Nuclear Genomes in New World Myotis Bats

The rapid diversification of Myotis bats into more than 100 species is one of the most extensive mammalian radiations available for study. Efforts to understand relationships within Myotis have primarily utilized mitochondrial markers and trees inferred from nuclear markers lacked resolution. Our current understanding of relationships within Myotis is therefore biased towards a set of phylogenetic markers that may not reflect the history of the nuclear genome. To resolve this, we sequenced the full mitochondrial genomes of 37 representative Myotis, primarily from the New World, in conjunction with targeted sequencing of 3648 ultraconserved elements (UCEs). We inferred the phylogeny and explored the effects of concatenation and summary phylogenetic methods, as well as combinations of markers based on informativeness or levels of missing data, on our results. Of the 294 phylogenies generated from the nuclear UCE data, all are significantly different from phylogenies inferred using mitochondrial genomes. Even within the nuclear data, quartet frequencies indicate that around half of all UCE loci conflict with the estimated species tree. Several factors can drive such conflict, including incomplete lineage sorting, introgressive hybridization, or even phylogenetic error. Despite the degree of discordance between nuclear UCE loci and the mitochondrial genome and among UCE loci themselves, the most common nuclear topology is recovered in one quarter of all analyses with strong nodal support. Based on these results, we re-examine the evolutionary history of Myotis to better understand the phenomena driving their unique nuclear, mitochondrial, and biogeographic histories

Comparison of the ruminal and fecal microbiotas in beef calves supplemented or not with concentrate.

Most of the research efforts involving the bovine gastrointestinal microbiota have focused on cattle's forestomach, particularly the rumen, so information concerning the bovine fecal microbiota is more scarce, especially in young beef cattle. The present study was performed to evaluate the ruminal and fecal microbiotas of beef calves as they reached the end of their nursing phase. A total of 18 Angus cow/calf pairs were selected and assigned to one of two treatment groups for the last 92 days of the calves' nursing period, as follows: 1) calves were supplemented with concentrate in a creep feeding system; or 2) control group with no supplementation of calves. After 92 days, ruminal and fecal samples were individually obtained from calves in both groups, and their microbiotas were evaluated using 16S rRNA gene sequencing. Ruminal samples were predominated by Prevotella (18 to 23% of the total bacterial abundance), regardless if calves received supplementation or not; however, in the feces, Prevotella was only the seventh most abundant genus (0.6 to 2.1% of total bacterial abundance). Both the rumen (P = 0.01) and the feces (P = 0.05) of calves that received supplementation had greater abundance of Firmicutes. In addition, calves that were supplemented had lower abundance of Fibrobacteres (P = 0.03) in their rumens. Regardless if the calves were supplemented or not, Faith's Phylogenetic Diversity index (P ≤ 0.007) and total concentration of short chain fatty acids (P < 0.001) were both greater in the rumen than in the feces of calves. In summary, the ruminal and fecal microbiotas of weanling beef calves were considerably distinct. Additionally, supplementation with creep feed caused some significant changes in the composition of the gastrointestinal microbiota of the calves, especially in the rumen, where supplementation caused an increase in Firmicutes and a decrease in abundance of Fibrobacteres

UCE_loci

Zip of .fasta and .incomplete files of UCE loci identified. Output from extract_fasta.sh and extract_fasta_empIns.sh scripts. Used as input for internal_trim.sh and internal_trim_empIns.sh scripts respectively

phyluce_workflow

Text file of script usage workflow of the analyses performed in PHYLUCE and RAxML

Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms

UCE_alignments

Zip of individual UCE loci alignments from MAFFT in nexus format, output of internal_trim.sh and internal_trim_empIns.sh scripts, for input into Gblocks

RAxML_alignment_input_&_Trees_output

Zip of UCE loci and character set files used as input for RAxML for both the "empirical" and "empirical + in silico" (empIns) data sets for the 60% data matrix. The output reconciled tree from RAxML for each data set