Molecular sequencing techniques help to understand microbial biodiversity with regard to species richness, assembly structure and function. In this context, available methods are barcoding, metabarcoding, genomics and metagenomics. The first two are restricted to taxonomic assignments, whilst genomics only refers to functional capabilities of a single organism. Metagenomics by contrast yields information about organismal and functional diversity of a community. However currently it is very demanding regarding labour and costs and thus not applicable to most laboratories. Here, we show in a proof-of-concept that computational approaches are able to retain functional information about microbial communities assessed through 16S rDNA (meta)barcoding by referring to reference genomes. We developed an automatic pipeline to show that such integration may infer preliminary or supplementary genomic content of a community. We applied it to two biological datasets and delineated significantly overrepresented protein families between communities.
Keller A, Horn H, Förster F, Schultz J. (2014) Computational integration of genomic traits into 16S rDNA microbiota sequencing studies. Gene. 549:1 186–191
Meta-barcoding of mixed pollen samples constitutes a suitable alternative to conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods, e.g. separate steps for amplification and sample indexing.
We thus developed a new primer-adapter design for high throughput sequencing with the Illumina technology that remedies these issues. It uses a dual-indexing strategy, where sample-specific combinations of forward and reverse identifiers attached to the barcode marker allow high sample throughput with a single sequencing run. It does not require further adapter ligation steps after amplification. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all samples together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, 2,000–3,000 high quality reads per sample were sufficient to assess the complete diversity of 95% of the samples. We were able to detect 650 different plant taxa in total, of which 95% were classified at the species level. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number of covered global plant species included in the database from 37,403 to 72,325 (93% increase).
This study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples.
Sickel W, M Ankenbrand, G Grimmer, A Holzschuh,S Härtel, J Lanzen, I Steffan-Dewenter, A Keller (2015) Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach. BMC Ecology 15: 20
The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, spe- cies-specific internal transcribed spacer two region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly cor- related with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.
Keller A, N Danner, G Grimmer, M Ankenbrand, K von der Ohe, W von der Ohe, S Rost, S Härtel, I Steffan-Dewenter (2014) Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples. Plant Biology, in press