Whole_genome_sequence_from_Cholera_patients_infused_onto_biocidal_and_treated_filter_papers-sc-4226
Whole_genome_sequence_from_Cholera_patients_infused_onto_biocidal_and_treated_filter_papers
To  facilitate better access to Vibrio cholera samples at epidemic sites we have been trialing sequencing whole genomes from culture/Stool soaked absorbent filter papers. We have performed preliminary experiments using Whatman FTA Elute Micro Cards (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and standard filter papers. The Whatman filter papers are design to kill the bacteria but protect the DNA. Through our collaborators we have been looking at bacterial viability of culture (Vibrio cholerae culture of density 5 x 10 ^9 cells/ml) spotted onto the filter papers and then: treated with ethanol; subjected to UV light; treated with bleach solution and then all dried at 37 C overnight. We have compared this to untreated filter papers which were simply dried at 37 C overnight. In all cases the cells are non-viable after having dried overnight. We have extracted the DNA from these cards and now want to determine the level of genome coverage we can generate from single filter papers.  This is an R & D project with up to 36 samples to sequence split in two phases.   Our project will comprise the following:  Phase 1 1) Currently we have 6x samples that have been titration of Vibrio gDNA load (5, 10, 20, 30 and 40 ng) to be sequenced using standard illumina libraries (R & D Team), together with gDNA directly extracted from the same culture, in order to find out the minimal amount of material, spots and cards that are necessary to collect samples in future from patients.  Phase 2 2) A study of cards spotted with stool and treated as before. This will include up to 20 samples. The aim is to assess gDNA extraction protocols and quantification as well as Illumina sequencing with standard libraries.   To note we have been discussing the use of Nextera XT kits for lesser gDNA loads.   Please note this is a R & D pilot project for which publication will be possible only if success and application to further future samples.   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/
This is a pilot R & D study to investigate the possibility of obtaining whole genome sequences from Cholera patients from which samples are infused onto biocidal and treated filter papers. The purpose is to facilitate better access to Vibrio cholera samples at epidemic sites. We have performed preliminary experiments using Whatman FTA Elute Micro Cards (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and standard filter papers. The Whatman filter papers are design to kill the bacteria but protect the DNA. In this pilot trial we will assess the viability of the  methodology (from total genomic  extraction to coverage and depth of genome of Vibrio cholera sequenced), in particular determining the minimum copy number of Vibrio cholera isolated from a card that will yield a succesful genome.