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Basics of Next Generation Sequencing

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NEXT GENERATION SEQUENCING – BASICS AND APPLICATIONS:

NEXT GENERATION SEQUENCING – BASICS AND APPLICATIONS SALAM DAYANANDA SINGH PhD SCHOLAR UNDER THE GUIDANCE: DR LISAM

WHAT IS NGS?:

WHAT IS NGS? High throughput sequencing Lower Cost Less time Parallel Sequencing process Sequence thousands of sequences at once

TECHNOLOGIES AND VENDORS:

TECHNOLOGIES AND VENDORS 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)

HOW ARE CAPILLARY SEQUENCING AND NGS ARE DIFFERENT?:

HOW ARE CAPILLARY SEQUENCING AND NGS ARE DIFFERENT? 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 : 1.2X10 6 reads < 75b/run

WHAT IS COMMON AMONG NGS TECHNOLOGIES?:

WHAT IS COMMON AMONG NGS TECHNOLOGIES? High Throughput Adapter ligation Requirement of relatively little input DNA Production of shorter read lengths(more convenient in downstream processing).

THREE MOST COMMONLY USED TECHNOLOGIES:

THREE MOST COMMONLY USED TECHNOLOGIES Roche 454 GS FLX sequencer (Pyrosequencing) Illumina genome analyzer (Sequencing by Synthesis) Applied Biosystems SOLiD sequencer (Sequencing by ligation)

ILLUMINA SEQUENCING WORKFLOW:

ILLUMINA SEQUENCING WORKFLOW

APPLICATIONS:

APPLICATIONS Mutation discovery Transcriptome Analysis – RNA- Seq Sequencing clinical isolates in strain-to-reference mechanisms. Enabling M etagenomics Defining DNA-Protein interactions – ChIP-Seq Discovering non-coding RNAs

MUTATION DISCOVERY:

MUTATION DISCOVERY 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.)

RNA-Seq:

RNA- Seq Massively Parallel Sequencing method for T ranscriptome analysis. mRNA (transcript) – cDNA – sequencing using Next Generation Short Read Sequencing technology. Reads are aligned to a reference genome and a T ranscriptome 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

Sequencing clinical isolates in strain-to-reference comparisons:

Sequencing clinical isolates in strain-to-reference comparisons 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.

Enabling Metagenomics:

Enabling Metagenomics 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 M etagenomic 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 M icrobiome .

Defining DNA-Protein Interactions:

Defining DNA-Protein Interactions DNA-Protein interactions – DNA packaging into histones Regulatory protein Binding Exploring Chromatin Packaging

Regulatory Protein Binding -- ChIP-Seq:

Regulatory Protein Binding -- ChIP-Seq 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.

Exploring Chromatin Packaging:

Exploring C hromatin Packaging 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.

Discovering Non-Coding RNAs (ncRNA):

Discovering Non-Coding RNAs ( ncRNA ) 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 .

What is after Ngs?!:

What is after Ngs ?! 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.

References::

References: 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

THANK YOU:

THANK YOU