An improved protocol for small RNA library construction using High Definition adapters

Next generation sequencing of small RNA (sRNA) libraries is widely used for studying sRNAs in various biological systems. However, cDNA libraries of sRNAs are biased for molecules that are ligated to adapters more or less efficiently than other molecules. One approach to reduce this ligation bias is to use a pool of adapters instead of a single adapter sequence, which allows many sRNAs to be ligated efficiently. We previously developed High Definition (HD) adapters for the Illumina sequencing platform, which contain degenerate nucleotides at the ligating ends of the adapters. However, the current commercial kits produced a large amount of 5’ adapter – 3’ adapter ligation product without the cDNA insert when HD adapters were used to replace the kit adapters. Here, we report a protocol to generate sRNA libraries using HD adapters with dramatically reduced adapter-adapter product. This protocol was developed from the procedure invented by Vaidyanathan et al. The libraries can be completed within two days and can be used for various biological and clinical samples. As examples for using this protocol, we constructed sRNA libraries using total RNA extracted from cultured mammalian cells and plant leaf tissue. The PCR products contained a very small amount of adapter-adapter product. Bioinformatic analysis of the sequencing data revealed sRNAs with diverse sequences and many different miRNA families

Draft Genome Sequences of Campylobacter jejuni Strains That Cause Abortion in Livestock.

Campylobacter jejuni is an intestinal bacterium that can cause abortion in livestock. This publication announces the public release of 15 Campylobacter jejuni genome sequences from isolates linked to abortion in livestock. These isolates are part of the 100K Pathogen Genome Project and are from clinical cases at the University of California (UC) Davis

How do you Play with a Robotic Toy Animal? A long-term study of Pleo

Pleo is one of the more advanced interactive toys currently available for the home market, taking the form of a robotic dinosaur. We present an exploratory study of how it was interacted with and reflected upon in the homes of six families during 2 to 10 months. Our analysis emphasizes a discrepancy between the participants’ initial desires to borrow a Pleo and what they reported later on about their actual experiences. Further, the data suggests an apparent tension between participants expecting the robot to work as a ‘toy’ while making consistent comparisons with real pet animals. We end by discussing a series of implications for design of this category of toys, in order to better maintain interest and engagement over time

Joint assembly and genetic mapping of the Atlantic horseshoe crab genome reveals ancient whole genome duplication

Horseshoe crabs are marine arthropods with a fossil record extending back approximately 450 million years. They exhibit remarkable morphological stability over their long evolutionary history, retaining a number of ancestral arthropod traits, and are often cited as examples of "living fossils." As arthropods, they belong to the Ecdysozoa}, an ancient super-phylum whose sequenced genomes (including insects and nematodes) have thus far shown more divergence from the ancestral pattern of eumetazoan genome organization than cnidarians, deuterostomes, and lophotrochozoans. However, much of ecdysozoan diversity remains unrepresented in comparative genomic analyses. Here we use a new strategy of combined de novo assembly and genetic mapping to examine the chromosome-scale genome organization of the Atlantic horseshoe crab Limulus polyphemus. We constructed a genetic linkage map of this 2.7 Gbp genome by sequencing the nuclear DNA of 34 wild-collected, full-sibling embryos and their parents at a mean redundancy of 1.1x per sample. The map includes 84,307 sequence markers and 5,775 candidate conserved protein coding genes. Comparison to other metazoan genomes shows that the L. polyphemus genome preserves ancestral bilaterian linkage groups, and that a common ancestor of modern horseshoe crabs underwent one or more ancient whole genome duplications (WGDs) ~ 300 MYA, followed by extensive chromosome fusion

Protocol for Metatranscriptomic analysis of Intestinal Microbiota

The objective of this publication is to provide the detailed protocol for metartancriptomisc studies of animal intestinal microbiota. The protocol describes isolation of high quality microbial community RNA from the mammalian intestinal content, subsequent mRNA enrichment, cDNA synthesis and sequencing. Twelve libraries were prepared, pooled in equimolar concentrations into a single library and sequenced on one GS Titanium 70×75 picotiter plate, following this protocol. The total number of reads obtained for 12 libraries was 1,155,062 (average 96,000 per library) and the combined size of 12 libraries was 521 million bases (average 43 million bases per library). The reported size of non-ribosomal RNA library fraction is ~15%, the fraction of non-ribosomal reads is ~17%. Hence we described a robust technique for metranscriptomic studies of animal intestinal microbiota. The double stranded cDNAs, prepared following this protocol, are suitable for pyrosequencing (454, Illumina), clone library construction or could be used to archive and store metaranscriptomic samples

Optimizing sequencing protocols for leaderboard metagenomics by combining long and short reads.

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing

Public health risk of Giardia and Cryptosporidium posed by reintroduction of beavers into Scotland

Following publication of ‘Scottish Beaver Trial Independent Public Health Monitoring 2009-2014 Report and Recommendations’ (Mackie, 2014), two pieces of complementary work were undertaken in parallel to assess the potential contribution of reintroduced beavers in Scotland to the public health burden of disease attributed to Giardia spp. and Cryptosporidium spp. parasites. The first, a risk assessment, addressing the question ‘What is the likelihood that re-introduced beavers will have a significant impact on the contamination of drinking water supplies with Cryptosporidium parvum and Giardia lamblia?’ (Appendix 1), was conducted by Scottish Government’s Centre of Expertise on Animal Disease Outbreaks (EPIC). This reviewed evidence from data and publications across the world, as well as evidence from the beaver trial and SNH’s Tayside beaver reports, and used this to assess the likely additional contribution of beavers to the risk associated with exposure to these parasites in Scotland. The second, ‘What is the likelihood that beavers will be an important source of contamination of drinking water supplies with Cryptosporidium parvum and Giardia intestinalis?’ (Appendix 2), was prepared by Health Protection Scotland (HPS), Scottish Parasite Diagnostic Reference Laboratory (SPDL) and Drinking Water Quality Regulator for Scotland (DWQR). This reviewed the diagnostics, surveillance and epidemiology of these infections in people in Scotland