High throughput sequencing
Parallel Sequencing process
Sequence thousands of sequences at once
Massively Parallel Signature Sequencing (Lynx
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
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
Requirement of relatively little input DNA
Production of shorter read lengths(more
convenient in downstream processing).
Discovery of mutations that determine
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
mRNA (transcript) – cDNA – sequencing using Next
Generation Short Read Sequencing technology.
Reads are aligned to a reference genome and a
Transcriptome map is constructed.
Does not require existing genomic sequence unlike
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
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
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
DNA-Protein interactions – DNA packaging into
Regulatory protein Binding
Exploring Chromatin Packaging
ChIP requires an antibody specific for the DNA
Protein DNA cross linker is added.
Cell lysis --- DNA fragmentation – Antibody
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
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
Elaine R. Mardis (2008) the impact of next-generation
sequencing technology on genetics. Cell vol.24
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