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Aim:The Purpose of the project is whole genome sequencing of bacterial DNA derived from single cells isolated from environmental samples, the genomic DNA amplified by multiple displacement amplification (MDA) prior to sequencing.For initial proof of concept studies cells will be isolated from bacterial cultures with a fully assembled genome sequence to enable examination of the coverage achieved from the amplified gDNA of a single cell.Methods:-Cell isolation was achieved by a combination of laser microbeam microdissection and micromanipulation.-Cell Lysis was mediated by incubation of samples at 95deg C .-MDA was performed using Genomiphi V2 mini kit from GE, following manufacturers basic protocol.-PCR of the 16S rRNA gene was used to identify the phylotype of isolated and amplified samples prior to submission for whole genome sequencing.Submitted samples:Proof of concept studies examining cells isolated from bacterial cultures aim to determine coverage and depth of sequence generated via the MDA reaction by comparing amplified samples to known sequencing data.The Actual study samples of uncultured bacterial phylotypes are processed to gain as clear a picture as possible of the genomic sequence of these previously uncharacterised phyla.Ultimate purpose of study:Data for uncultured bacteria sequenced as part of this project will feed directly into the MetaHit consortium project for the study of the microbiota of the human gut.. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ |