Single Bacterial Cell Whole Genome Amplification-sc-2010-03-22
Single Bacterial Cell Whole Genome Amplification
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/
In order to facilitate sequencing of uncultured phylotypes from the human gut microbiota it was first necessary to isolate individual bacterial cells via Laser microdissection microscopy. Isolated individual cells were lysed by heat treatment in a stabilising sample buffer. Genomic DNA released underwent Multiple Displacement Amplification (MDA) to amplify the whole genome, the Genomiphi V2 mini kit was used for this reaction. 16S rRNA gene PCR was used to determine the phylotype of the amplification. Samples of interest were submitted for illumina whole genome sequencing with the aim of generating the first genome sequence for the uncultured phylotypes isolated. The resulting sequence data will feed into the MetaHit consortium study.