Project description:Comparison of faecal flora of three healthy individuals and a patient suffering from Ulcerative Colitis during disease and remission states. Faecal samples were taken and frozen at -80 within one hour.
Project description:PCR read-off microarrays: The ability to efficiently and economically generate libraries of defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, such as protein engineering and aptamer library screening, but also in applications associated with encoding and tagging. By this approach DNA microarrays were used to allow the linear amplification off immobilized DNA sequences on an array followed by PCR amplification. Arrays of increasing sophistication (1; 10; 3,875; 10,000 defined sequences) were used to validate the process, with sequences verified by selective hybridization to a complementary DNA microarray and DNA sequencing. This technique offers an economical and efficient way of producing hundreds to thousands of specific DNA libraries, while the DNA-arrays can be used as factories allowing specific DNA oligonucleotide pools to be generated with or without masking.
Project description:To further decipher the alteration of gene expression profile of irradiated mice with or without faecal microbiota transplantation (FMT), we performed FMT for 10 days following total body irradiaton (6.5 Gy gamma ray). Twenty-one days after irradiation, the mice were euthanized and the small intestine tissues excised. Overall design: The male (and female) mice were separated into four groups randomly, The mice in Group A were treated with saline as negative control; the mice in Group B were treated with sex-matched FMT; the mice in Group C were treated with sex-mismatched FMT and the mice in Group D were sex-mixed (the weight of male and female stool was 1:1) FMT. FMTs were performed for 10 days following total body irradiaton (6.5 Gy gamma ray ). Twenty-one days after irradiation, the mice were euthanized and the small intestine tissues excised.
Project description:We assayed leukocyte global gene expression for a prospective discovery cohort of 106 adult patients admitted to UK intensive care units with sepsis due to community acquired pneumonia or faecal peritonitis. We assigned all samples to sepsis response signature groups after performing unsupervised analysis of the transcriptomic data.
Project description:We assayed leukocyte global gene expression for a prospective validation cohort of 221 adult patients admitted to UK intensive care units with sepsis due to community acquired pneumonia or faecal peritonitis. 10 samples from patients scheduled for elective cardiac surgery were also assayed as non-septic controls. We assigned all samples to sepsis response signature groups after performing unsupervised analysis of the transcriptomic data.
Project description:Next-generation sequencing has been widely used for the genome-wide profiling of histone modifications, transcription factor binding and gene expression through chromatin immunoprecipitated DNA sequencing (ChIP-seq) and cDNA sequencing (RNA-seq). Here, we describe a versatile library construction method that can be applied to both ChIP-seq and RNA-seq on the widely used Illumina platforms. Standard methods for ChIP-seq library construction require nanograms of starting DNA, substantially limiting its application to rare cell types or limited clinical samples. By minimizing the DNA purification steps that cause major sample loss, our method achieved a high sensitivity in ChIP-seq library preparation. Using this method, we achieved the following: (1) generated high-quality epigenomic and transcription factor-binding maps using ChIP-seq for murine adipocytes; (2) successfully prepared a ChIP-seq library from as little as 25 pg of starting DNA; (3) achieved paired-end sequencing of the ChIP-seq libraries; (4) systematically profiled gene expression dynamics during murine adipogenesis using RNA-seq; and (5) preserved the strand specificity of the transcripts in RNA-seq. Given its sensitivity and versatility in both double-stranded and single-stranded DNA library construction, this method has wide applications in genomic, epigenomic, transcriptomic and interactomic studies. Pre-adipocytes and mature adipocytes were collected. Their chromatin and RNA were subjected to ChIP and mRNA extraction. Sequencing libraries from ChIP DNA or mRNA were generated following either standard protocols or TELP method. The quality and features of TELP libraries were proved and demonstrated in comparison with standard libraries or other published data.
Project description:Deep sequencing of transcriptomes allows quantitative and qualitative analysis of many RNA species in a sample, with parallel comparison of expression levels, splicing variants, natural antisense transcripts, RNA editing and transcriptional start and stop sites the ideal goal. By computational modeling, we show how libraries of multiple insert sizes combined with strand-specific, paired-end (SS-PE) sequencing can increase the information gained on alternative splicing, especially in higher eukaryotes. Despite the benefits of gaining SS-PE data with paired ends of varying distance, the standard Illumina protocol allows only non-strand-specific, paired-end sequencing with a single insert size. Here, we modify the Illumina RNA ligation protocol to allow SS-PE sequencing by using a custom pre-adenylated 3’ adaptor. We generate parallel libraries with differing insert sizes to aid deconvolution of alternative splicing events and to characterize the extent and distribution of natural antisense transcription in C. elegans. Despite stringent requirements for detection of alternative splicing, our data increases the number of intron retention and exon skipping events annotated in the Wormbase genome annotations by 127 % and 121 %, respectively. We show that parallel libraries with a range of insert sizes increase transcriptomic information gained by sequencing and that by current established benchmarks our protocol gives competitive results with respect to library quality. Sequencing of mRNA from C. elegans with libraries of four differing insert sizes