High-throughput sequencing of African chikanda cake highlights conservation challenges in orchids

Source at https://doi.org/10.1007/s10531-017-1343-7 .Chikanda is a traditional dish made with wild-harvested ground orchid tubers belonging to three orchidioid genera, Disa, Satyrium and Habenaria, all of which are CITES appendix II-listed. Identification of collected orchid tubers is very difficult and documentation of constituent species in prepared chikanda has hitherto been impossible. Here amplicon metabarcoding was used in samples of six prepared chikanda cakes to study genetic sequence diversity and species diversity in this product. Molecular operational taxonomic unit identification using similarity-matching reveals that species of all three genera were present in the chikanda samples studied. Disa was present in all of the samples, Satyrium in five out of six and Habenaria in one of the samples, as well as a number of other plants. The fact that each sample contained orchids and the presence of a wide variety of species from all genera in this traditional dish raise serious concerns about the sustainability of this trade and the future of wild orchid populations in the main harvest areas. This proof-of-concept study shows that Ion-Torrent PGM is a cost-effective scalable platform for metabarcoding using the relatively long nrITS1 and nrITS2 regions. Furthermore, nrITS metabarcoding can be successfully used for the detection of specific ingredients in a highly-processed food product at genus level, and this makes it a useful tool in the detection of possible conservation issues arising from commercialized trade or processed plant products

Microbiome and environment explain the absence of correlations between consumers and their diet in Bornean microsnails

Classical ecological theory posits that species partition resources such that each species occupies a unique resource niche. In general, the availability of more resources allows more species to co‐occur. Thus, a strong relationship between communities of consumers and their resources is expected. However, correlations may be influenced by other layers in the food web, or by the environment. Here we show, by studying the relationship between communities of consumers (land snails) and individual diets (from seed plants), that there is in fact no direct, or at most a weak but negative, relationship. However, we found that the diversity of the individual microbiome positively correlates with both consumer community diversity and individual diet diversity in three target species. Moreover, these correlations were affected by various environmental variables, such as anthropogenic activity, habitat island size, and a possibly important nutrient source, guano runoff from nearby caves. Our results suggest that the microbiome and the environment explain the absence of correlations between diet and consumer community diversity. Hence, we advocate that microbiome inventories are routinely added to any community dietary analysis, which our study shows can be done with relatively little extra effort. Our approach presents the tools to quickly obtain an overview of the relationships between consumers and their resources. We anticipate our approach to be useful for ecologists and environmentalist studying different communities in a local food web

Exon expression arrays as a tool to identify new cancer genes

Background: Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples. Principal Findings: As a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs). Conclusions: The ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes

The mitochondrial genome of Nemalecium lighti (Hydrozoa, Leptothecata)

The hydrozoan species Nemalecium lighti (Hargitt, 1924) is widely distributed in tropical marine waters around the world. Here we report the complete linear mitochondrial genome of N. lighti from Sint Eustatius (Lesser Antilles). The mitochondrial genome with a length of 14,320 bp encodes for 13 protein-coding genes, two tRNA genes, and two rRNA genes. Gene arrangement differs from that found in other species of the same taxonomic order and a phylogenetic analysis shows that based on mitochondrial genes, N. lighti clusters outside of the Leptothecata, rendering the order paraphyletic

BWA alignments in SAM format

BWA alignments in SAM forma

Data from: Parallel tagged amplicon sequencing of transcriptome-based genetic markers for Triturus newts with the Ion Torrent next-generation sequencing platform

Next-generation sequencing is a fast and cost-effective way to obtain sequence data for non-model organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next we test the utility of our markers. BAPS allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. NewHybrids, a hybrid index and HIest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus

Gender-based variation in the diet of the Pond bat using metabarcoding of dung pellets

DNA extraction from faecal pellets We analysed a total of 253 faecal pellets with DNA metabarcoding. A total of 93 pellets did not yield results (31 males and 62 females) and were excluded from further analysis. The remaining pellets were 95 females and 65 males. Details of all pellets analysed are provided in Supplementary Data SD2b. The pellets were ground to a fine powder in liquid nitrogen with a mortar and pestle using the protocol of the commercial Qiagen QIAamp DNA Stool Mini Kit in a special Ancient DNA facility dedicated to work with samples with degraded DNA and following established protocols to avoid contamination such as the inclusion of extraction blanks (Cooper and Poinar 2000). Subsequently, aliquots of each extraction were further purified using Promega PCR purification columns. PCR amplification of COI and 16S mini-barcodes and library preparation Amplifications of the ~313 bp long mitochondrial COI mini-barcoding marker were performed using forward primer ZBJ-ArtF1c 5’-AGATATTGGAACWTTATATTTTATTTTTGG-3’ and reverse primer ZBJ-ArtR2c 5’- WACTAATCAATTWCCAAATCCTCC-3’ (Zeale et al. 2011). The ~157 bp long mitochondrial 16S barcoding marker was amplified using the forward primer P7_FO-16S 5’- RGACGAGAAGACCCTATARA-3’ and P7_R0-16S 5’-ACGCTGTTATCCCTAARGTA-3’ (Elbrecht et al. 2016). Primers were labelled for DNA metabarcoding with IonExpress labels. The PCR was carried out in 30 microliter reactions containing 0.20 µl Qiagen taq 5u/µl, 3 µl 10x Qiagen buffer, 2 µl 2,5mM dNTP’s, 0,5 µl 10 µM forward primer, 0,5 µl 10 µM reverse primer, 1,5 µl 25mM MgCl2, 0,5 µl 10 mg/ml BSA, 19.80 µl MiliQ and 2 µl template. Amplifications were performed using the following PCR programme: 5 min denaturation at 95°C followed by 40 cycles of 20 seconds denaturation at 95°C, 20 seconds annealing at 50°C and 1-minute elongation at 72°C. Final elongation was conducted at 72°C for 7 minutes on a C1000 Biorad PCR machine. Ion Torrent sequencing Primer dimer and other contaminants were removed by using 0.9x Ampure XP beads (Agencourt) to which the PCR products were bound. The beads were washed with 150 microliter 70% EtOH twice and resuspended in 20 microliter TE buffer. Cleaned PCR products were quantified using an Agilent 2100 Bioanalyzer DNA High sensitivity chip. An equimolar pool was prepared of the amplicon libraries at the highest possible concentration. This equimolar pool was diluted according to the calculated template dilution factor to target 10-30% of all positive Ion Sphere Particles. Template preparation and enrichment was carried out with the Ion One Touch 200 Template kit with use of the Ion One Touch System, according to the manufacturer's protocol. The quality control of the Ion One Touch 200 Ion Sphere Particles was done with the Ion Sphere Quality Control kit using a Life Qubit 2.0. The enriched Ion Spheres were prepared for sequencing on a Personal Genome Machine (PGM) with the Ion PGM 200 Sequencing kit as described in the protocol and deposited on an Ion-314 chip (520 cycles per run) in three consecutive sequencing runs. Sequence analysis and data filtering Reads obtained from Ion Torrent sequencing were automatically sorted into separate sequence files based on the MID labels by the Ion Torrent software. The reads were further processed with PRINSEQ (version 0.20.3, Schmieder and Edwards 2011) with the following settings: a minimum read length of 100 bp, trimming to 140 bp, minimum mean quality of Q24 per read, additional trimming of '3 end bases with a Q lower than 24 and removal of full duplicate sequences. Filtered reads were clustered into Operational Taxonomic Units (OTUs) defined by a sequence similarity of at least 97% using CD-HIT-EST (Li and Godzik 2006). Singletons were omitted. For each cluster the representative sequences were BLASTed with the NCBI-blast+ software package (version 2.2.28+, Camacho et al. 2009) against either the NCBI GenBank nucleotide database or a custom database containing all Arthropod sequences located on the Barcode of Life Database (Ratnasingham and Hebert 2007). BLAST hits were filtered according to the following criteria: minimum hit length of a 100 bp, minimum hit similarity of 97% and a maximum e-value of 0.05. Reference databases of Dutch species such as http://www.nederlandsesoorten.nl/ were used to check if a species had been recorded for the Netherlands. All species not (yet) known for The Netherlands were reduced to genus level or to the family level if the genus is also unknown to occur

Microbiota in Dung and Milk Differ Between Organic and Conventional Dairy Farms

Organic farming is increasingly promoted as a means to reduce the environmental impact of artificial fertilizers, pesticides, herbicides, and antibiotics in conventional dairy systems. These factors potentially affect the microbial communities of the production stages (soil, silage, dung, and milk) of the entire farm cycle. However, understanding whether the microbiota representative of different production stages reflects different agricultural practices – such as conventional versus organic farming – is unknown. Furthermore, the translocation of the microbial community across production stages is scarcely studied. We sequenced the microbial communities of soil, silage, dung, and milk samples from organic and conventional dairy farms in the Netherlands. We found that community structure of soil fungi and bacteria significantly differed among soil types, but not between organic versus conventional farming systems. The microbial communities of silage also did not differ among conventional and organic systems. Nevertheless, the dung microbiota of cows and the fungal communities in the milk were significantly structured by agricultural practice. We conclude that, while the production stages of dairy farms seem to be disconnected in terms of microbial transfer, certain practices specific for each agricultural system, such as the content of diet and the use of antibiotics, are potential drivers of shifts in the cow’s microbiota, including the milk produced. This may reflect differences in farm animal health and quality of dairy products depending on farming practices

Bacteriën en schimmels op melkveebedrijf in beeld

De samenstelling van de bacterie- en schimmelgemeenschap als geheel is specifiek voor elk onderdeel van de kringloop op een melkveebedrijf. Dat laat een verkennend onderzoek gefinancierd door de Triodos Foundation zien. Zo is de bacterie- en schimmelgemeenschap in de bodem heel anders dan die in de mest. In de mest en melk was er een verschil te zien tussen gangbare en biologische bedrijven