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Next-generation sequencing and PBRC

Next-generation sequencing and PBRC. Next Generation Sequencer Applications. DeNovo Sequencing Resequencing, Comparative Genomics Global SNP Analysis Gene Expression Analysis Methylation Studies ChIP Sequencing-transcription factors, histones, polymerases

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Next-generation sequencing and PBRC

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  1. Next-generation sequencing and PBRC

  2. Next Generation Sequencer Applications • DeNovo Sequencing • Resequencing, Comparative Genomics • Global SNP Analysis • Gene Expression Analysis • Methylation Studies • ChIP Sequencing-transcription factors, histones, polymerases • Transcriptome Analysis-splicing, UTRs, cSNPs, nested transcripts • MicroRNA Discovery and quantitation • Metagenomics, Microbial diversity • Copy number variation • Chromosomal aberrations • Gene regulation studies

  3. AB SOLiD Ligation sequencing

  4. How many sequence tags* do I need for my gene expression application? • SAGE/CAGE – 2-5 million mappable • miRNA – 10 million mappable • ChIP Seq—10-20 million mappable • Whole Transcriptome from polyA RNA – 40-50 million mappable • Whole Transcriptome from rRNA depleted - >50 million mappable • Whole Transcriptome for Allele Specific Expression - >>50 million mappable SOLiD™ 4 generates >1.4 billion mappable sequences/run (2 slides) Libraries can be multiplexed to decrease the cost/sample according to the application and number of sequences needed. *For human/mouse sized genomes; smaller organisms require fewer sequence tags.

  5. SAGE Sequencing vs. Microarray

  6. Bioinformatics: Geospiza Primary Data Analysis - Images to bases Instrument-specific Sequences + Quality values RunQuality Ref Seq + Alignment Assembly, De Novo Secondary Data Analysis – Bases to alignments/contigs • Applications • Tag Profiling • Small RNA Analysis • Transcriptome seq. • ChIP-Seq • Methylation Analysis • Resequencing • De novo assembly • Algorithms • Eland • Maq • SOAP • Velvet • Newbler • Mapreads • Others … Sample/Library Quality One or more Data sets Tertiary Data Analysis – Experiment Specific • Differential expression • Methylation sites • Binding sites • Gene association • Genomic structure Discovery

  7. Next-gen sequencing: applications • Genome analysis: basic and translational research • Genetics of disease – new frontiers • Exomeresequencing: confirmation of GWAS • Genome sequence as diagnostic tool • Genetic counseling • Epigenome analysis: basic research; biomarkers • Analyses of DNA methylation, transcription factors, histone modifications, non-coding RNA • Epigenomic biomarkers of disease • Gene expression analysis: basic research; diagnostics & biomarkers • Whole transcriptome: all transcribed sequences in a cell • SAGE analysis: expression of known genes • Small RNA: microRNA as regulators of biology • Genotype to phenotype: a new frontier • Pathology: systems biology • Diagnosis: data filtering • Personalized Genomic Medicine: Treatment recommendations

  8. Next-gen sequencing: challenges • Rapid growth in methodology • Technology and equipment changes & upgrades • High demands on informatics: • Staff • Software • Computational resources • New ways of handling data needed: • Interpretation • Publication • Storage

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