Dual-index pollen meta-barcoding
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REPOSITORIES: ENA
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Project description: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.
| S-EPMC4509727 | biostudies-literature
Project description:We isolated tricellular pollen (TCP) and pollen mother cells (PMC) of rice using laser microdissection, and did microarray analysis with Agilent 44k rice array.
2010-07-31 | GSE17098 | GEO
Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis.
2009-07-28 | GSE17343 | GEO
Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis. Experiment Overall Design: Pollen and pollen tubes were collected as described in the protocols section for RNA extraction and hybridization on Affymetrix ATH1 Genechip microarrays.
2009-08-15 | E-GEOD-17343 | biostudies-arrayexpress