ABSTRACT: Bacterial 16S amplicon 454 pyrosequencing of environmental samples (vertebrate faeces, sediment, soil, surface water) to assess the potential of next generation sequencing methods for health related microbial water quality monitoring
Project description:The presence of numerous chemical contaminants from industrial, agricultural, and pharmaceutical sources in water supplies poses a potential risk to human and ecological health. Current chemical analyses suffer from limitations including chemical coverage and high cost, and broad-coverage in vitro assays such as transcriptomics may further improve water quality monitoring by assessing a large range of possible effects. Here, we used high-throughput transcriptomics to assess the activity induced by field-derived water extracts in MCF7 breast carcinoma cells.
Project description:Contaminated aquifer (Dusseldorf-Flinger, Germany) templates extracted from 5 sediment depths ranging between 6.4 and 8.4 m below ground and over 3 years of sampling were amplified for amplicon pyrosequencing using the primers Ba27f (5’-aga gtt tga tcm tgg ctc ag-3’) and Ba519r (5’- tat tac cgc ggc kgc tg-3’), extended as amplicon fusion primers with respective primer A or B adapters, key sequence and multiplex identifiers (MID) as recommended by 454/Roche. Amplicons were purified and pooled as specified by the manufacturer. Emulsion PCR (emPCR), purification of DNA-enriched beads and sequencing run were performed following protocols and using a 2nd generation pyrosequencer (454 GS FLX Titanium, Roche) as recommended by the developer. Quality filtering of the pyrosequencing reads was performed using the automatic amplicon pipeline of the GS Run Processor (Roche), with a slight modification concerning the valley filter (vfScanAllFlows false instead of TiOnly) to extract the sequences. Demultiplexed raw reads were furhter trimmed for quality and lenght (>250 bp).
Project description:High-throughput sequencing of Arabidopsis thaliana endogenous small RNAs by 454 pyrosequencing. Keywords: high-throughput sequencing
Project description:Contaminated aquifer (Dusseldorf-Flinger, Germany) templates extracted from 5 sediment depths ranging between 6.4 and 8.4 m below ground and over 3 years of sampling were amplified for amplicon pyrosequencing using the primers Ba27f (5’-aga gtt tga tcm tgg ctc ag-3’) and Ba519r (5’- tat tac cgc ggc kgc tg-3’), extended as amplicon fusion primers with respective primer A or B adapters, key sequence and multiplex identifiers (MID) as recommended by 454/Roche. Amplicons were purified and pooled as specified by the manufacturer. Emulsion PCR (emPCR), purification of DNA-enriched beads and sequencing run were performed following protocols and using a 2nd generation pyrosequencer (454 GS FLX Titanium, Roche) as recommended by the developer. Quality filtering of the pyrosequencing reads was performed using the automatic amplicon pipeline of the GS Run Processor (Roche), with a slight modification concerning the valley filter (vfScanAllFlows false instead of TiOnly) to extract the sequences. Demultiplexed raw reads were furhter trimmed for quality and lenght (>250 bp). 15 samples examined in total from important plume zones of the aquifer sampled in Feb. 2006, Sep. 2008 and Jun. 2009 (5 every year of sampling).
Project description:Methods currently available to estimate the post-mortem submerged interval (PMSI) of cadavers in water suffer from poor accuracy, being mostly based on morphological examination of the remains. Proteins present within bones have recently attracted more attention from researchers interested in the estimation of the post-mortem interval (PMI) in terrestrial environments. Despite the great potential of proteomic methods for PMI estimation, their application to aquatic environments has not yet been explored. In this study, we examined whether four different types of aquatic environment (tap water, saltwater, pond water and chlorinated water) affected the proteome of mice bones with increasing PMSIs (from zero to three weeks).
Project description:Mustard (Brassica juncea) was tested for Turnip mosaic virus infection. Small RNA of the plant was extracted and converted to DNA according to Ho, T., et al. (2006) Journal of Virological Methods 136:217-223, with primers modified to contain 454 adapter nucleotide sequences. The DNA then passed quality control through Bioanalyzer and Nanodrop before sequenced by 454 Life Sciences. Keywords: siRNA
Project description:Characterization of gene expression levels with RNA-seq was performed on self-renewing (SR) and senescent (SEN) human adult adipose derived mesenchymal stem cells (hADSCs) using the Roche 454 pyrosequencing platform.
Project description:The small RNAs of Selaginella moellendorffii were sampled by direct pyrosequencing (454 Life Sciences). These data represent microRNAs, siRNAs, and other expressed small RNAs of ~18-~28 nts. in length. Keywords: Small RNA sequencing - high throughput
Project description:Mustard (Brassica juncea) was tested for Turnip mosaic virus infection. Small RNA of the plant was extracted and converted to DNA according to Ho, T., et al. (2006) Journal of Virological Methods 136:217-223, with primers modified to contain 454 adapter nucleotide sequences. The DNA then passed quality control through Bioanalyzer and Nanodrop before sequenced by 454 Life Sciences. Keywords: siRNA One sample analyzed by 454 high-throughput sequencing technology
