Next generation sequencing

 High throughput sequencing
 Lower Cost
 Less time
 Parallel Sequencing process
 Sequence thousands of sequences at once

 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)

 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

 High Throughput
 Adapter ligation
 Requirement of relatively little input DNA
 Production of shorter read lengths(more
convenient in downstream processing).

 Roche 454 GS FLX sequencer
 (Pyrosequencing)
 Illumina genome analyzer
 (Sequencing by Synthesis)
 Applied Biosystems SOLiD sequencer
 (Sequencing by ligation)

 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

 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.)

 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

 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.

 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.

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

 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.

 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.

 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.

 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.

 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

Next generation sequencing

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