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Next generation sequencing

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  • 1. UNDER THE GUIDANCE: DR LISAM
  • 2.  High throughput sequencing  Lower Cost  Less time  Parallel Sequencing process  Sequence thousands of sequences at once
  • 3.  Massively Parallel Signature Sequencing (Lynx Therapeutics)  Polony Sequencing (Agencourt Biosciences)  454 Pyrosequencing (454 Life Sciences)  Illumina (Solexa) sequencing  SOLiD Sequencing (Applied Bio-systems)  Ion Semiconductor sequencing (Ion Torrent Systems Inc.)  DNA Nanoball (Complete Genomics)  Heli-oscope Single Molecule Sequencing  Single Molecule SMRT Sequencing (Pacific Biosciences)
  • 4.  The ability to process millions of sequence reads in parallel rather than 96 at a time.  NGS fragment libraries do not need vector based cloning and E. coli based amplification stages used in capillary sequencing.  Shorter Read Lengths.  Capillary sequencing – 96 wells, NGS – 10 million wells  High throughput : Sanger: 96 reads < 800-1000b/run Solexa: 1.2X106 reads < 75b/run
  • 5.  High Throughput  Adapter ligation  Requirement of relatively little input DNA  Production of shorter read lengths(more convenient in downstream processing).
  • 6.  Roche 454 GS FLX sequencer  (Pyrosequencing)  Illumina genome analyzer  (Sequencing by Synthesis)  Applied Biosystems SOLiD sequencer  (Sequencing by ligation)
  • 7.  Mutation discovery  Transcriptome Analysis – RNA-Seq  Sequencing clinical isolates in strain-to-reference mechanisms.  Enabling Metagenomics  Defining DNA-Protein interactions – ChIP-Seq  Discovering non-coding RNAs
  • 8.  Discovery of mutations that determine phenotypes.  Conventional Approach – PCR amplified – Capillary sequencing – alignment/detection.  Whole genome resequencing is faster and less expensive using NGS.  E.g. Discovery of SNP in C. elegans required only a single run of Illumina Sequencer. (Hiller et.al.)
  • 9.  Massively Parallel Sequencing method for Transcriptome analysis.  mRNA (transcript) – cDNA – sequencing using Next Generation Short Read Sequencing technology.  Reads are aligned to a reference genome and a Transcriptome map is constructed.  Advantages :  Does not require existing genomic sequence unlike hybridization.  Low background noise  High resolution – up to 1 bp (identification of SNP)  High throughput, low cost
  • 10.  Even though complete genome sequence are available for disease causing microbes, continuous evolution by mutation and sequence exchange.  The depth of sampling of NGS helps greatly in identification of rare VARIANTS in the clinical strain isolates.  This is not possible in sequencing PCR products which is commonly done in a clinical diagnostic setting, because the low signal strength from variant nucleotides would not be detectable on a capillary sequencer.  The cloning bias is eliminated.  Improve diagnostics, monitoring and treatments.
  • 11.  Metagenomics – sequencing of DNA of uncultured/unpurified microbial population followed by bioinformatics based analysis by comparison.  Associated cost of capillary sequencing remains very high.  Elimination of Metagenomic signatures from certain microbial sequences that are not carried stably by E.coli. during cloning.  Characterizations of the microbial census of the human and mouse intestinal flora and the oral cavity Microbiome.
  • 12.  DNA-Protein interactions – DNA packaging into histones  Regulatory protein Binding  Exploring Chromatin Packaging
  • 13.  ChIP requires an antibody specific for the DNA binding protein.  Protein DNA cross linker is added.  Cell lysis --- DNA fragmentation – Antibody Immunoprecipitation.  Crosslinking reversal or southern blotting or qPCR  ChIP-Seq --- simply make an adaptor ligated library of the released immunoprecipitated fragments and sequence them en masse.  High coverage and higher resolution.  NRSF and STAT1 transcription factors.
  • 14.  Genomic DNA packaging into histones – availability of genes for transcription.  ChIP-Seq to compare histone methylations at promoter regions to check gene expression levels.  In a study, 20 histones, one histone variant (H2A.Z), RNA Polymerase II and insulator binding protein.  Result: Changes in Chromatin state at specific promoters reflect changes in gene expression they control.
  • 15.  ncRNAs– regulatory RNA molecules.  Prediction of precursor and sequences of ncRNA by in silico methods is of limited use.  Examines the potential for secondary structure formation, putative genomic identification and regulatory molecules.  Identification of 21-U -RNAs in C.elegans.
  • 16.  Third generation (Next-Next Generation) Sequencing.  Variations in sequences of human genome (about 5% considering the allele variation) is found using NGS.  A pilot project for determination of additional Human Genome sequences.
  • 17.  Elaine R. Mardis (2008) the impact of next-generation sequencing technology on genetics. Cell vol.24 No.3,133-14  Jorge S Reis-Filho (2010): Next-Generation Sequencing, Breast Cancer Research 2010, 11(Suppl 3)  Elaine R. Mardis (2009): Next-Generation Sequencing Methods. Annu. Rev. Genomics hum genet. 9:387-402  Some websites

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