Chem 395 Bioanalytical Chemistry
Storrs, Connecticut
February 8, 2011
Modern Genomic Analysis Methods
Janet Hager
Translational Genomics Core
Department of Genetics and Developmental Biology
University of Connecticut Health Center
Genomic Resources at the UCHC
Translational Genomics Core
Department of Genetics and Developmental Biology
Cell
4 and Genome Sciences Center - 400 Farmington Avenue
Sample QC
Agilent Bioanalyzer 2100
Nanodrop 1000Ill
Microarrays
Illumina BeadStation and BeadChip: Gene Expression and Infinium and
GoldenGate genotyping
Illumina BeadXpress: Custom Veracode genotyping panels
Affymetrix Chip and Fluidics Station: Gene Expression, CNV, genotyping
Deep Sequencing
Illumina Genome Analyzer IIx
Ilumina HiSeq2000
Illumina Next Gen Sequencing Method
The basic goal for sample prep in all 2nd
generation next gen sequencing are the same:
 Generate large numbers of unique
"polonies" (polymerase generated colonies) or
“clusters” that can be simultaneously
sequenced.
 These parallel reactions occur on the
surface of a "flow cell" (basically a water-tight
microscope slide) which provides a large
surface area for many thousands of parallel
chemical reactions.
Illumina Next Gen Sequencing Method
Step 1: Sample Preparation
 DNA sample of interest is sheared to appropriate size
(average ~800bp) using a compressed air device known as
a nebulizer.
 Ends of the DNA are polished, and two unique adapters
are ligated to the fragments.
 Ligated fragments of the size range of 150-200bp are
isolated via gel extraction and amplified using limited
cycles of PCR.
 Complete detailed protocols for DNA, RNA and small
RNA library preparation
 This process is a fairly straightforward multi-step
molecular biology process
Illumina Next Gen Sequencing Method
Steps 2-6: Cluster Generation by Bridge Amplification
 454 and ABI SOLiD methods use a bead-based emulsion PCR to
generate "polonies”
 Illumina uses a unique "bridged" amplification reaction that occurs
on the surface of the flow cell.
 Flow cell surface is coated with single stranded oligonucleotides
that correspond to the sequences of the adapters ligated during the
sample preparation stage.
 Single-stranded, adapter-ligated fragments are bound to the
surface of the flow cell exposed to reagents for polyermase-based
extension.
Illumina Next Gen Sequencing Method
Illumina Next Gen Sequencing Method
Illumina Next Gen Sequencing Method
Steps 2-6: Cluster Generation by Bridge Amplification
 Repeated denaturation and extension results in localized
amplification of single molecules in millions of unique locations
across the flow cell surface.
 This process occurs in what is referred to as Illumina's "cluster
station", an automated flow cell processor.
 Priming occurs as the free/distal end of a ligated fragment
"bridges" to a complementary oligo on the surface.
Illumina Next Gen Sequencing Method
Illumina Next Gen Sequencing Method
Illumina Next Gen Sequencing Method
Steps 7-12: Sequencing by Synthesis
 Flow cell containing millions of unique clusters is now loaded into the
sequencer for automated cycles of extension and imaging.
 The first cycle of sequencing consists first of the incorporation of a
single fluorescent nucleotide, followed by high resolution imaging of the
entire flow cell.
 Images represent the data collected for the first base. Any signal above
background identifies the physical location of a cluster (or polony), and
the fluorescent emission identifies which of the four bases was
incorporated at that position.
 This cycle is repeated, one base at a time, generating a series of images
each representing a single base extension at a specific cluster.
 Base calls are derived with an algorithm that identifies the emission
color over time. Illumina reads range from 36-150 bases.
Illumina Next Gen Sequencing Method
Structure of reversible
terminator
Illumina Next Gen Sequencing Method
Illumina Genome Analyzer IIx
The Genome AnalyzerIIx has a
single 8 sample flowcell = 8
samples per run
• up to 2 x 150 bp read lengths
• up to 640 million paired-end
reads per flow cell,
• enabling a broad range of highthroughput sequencing
applications:
• RNA-seq: expression, isoform,
splice variants
• small RNA-seq: microRNA, ncRNA
• Whole genome-seq: SNP variants,
CNV
• Exome-seq: expression, variants
• ChIP-seq: transcription factor
regulatory sites
Illumina Genome Analyzer IIx
Illumina Genome Analyzer IIx Optical Path
Illumina Genome Analyzer IIx
Flow cell imaging
Illumina Genome Analyzer IIx
Illumina HiSeq 2000
Illumina HiSeq 2000
Illumina HiSeq 2000
Illumina HiSeq 2000
Illumina HiSeq 2000
Two flow cell capacity with 8 sample lanes per flow cell = 16 samples per run
Other 2nd Generation Sequencing
Platforms
• ABI SOLiD – sequencing by ligation; two base
calling; short read.
• Roche 454 – emulsion PCR; pyrosequencing; long
read
• Life Technologies Ion Torrent PGM – Personal
Genome Machine. Ion Torrent uses the simplest
sequencing chemistry including natural nucleotides, no
enzymatic cascade, no fluorescence, no
chemiluminescence, no optics, no light: The Chip is the
Machine.TM
• Helicos – Single molecule sequencing
Ion Torrent
• Ion Torrent uses a high-density array of micro-machined wells to perform this biochemical
process in a massively parallel way.
• Each well holds a different DNA template generated by emulsion PCR. Beneath the wells is
an ion-sensitive layer and beneath that a proprietary Ion sensor.
• The Ion Personal Genome Machine (PGM™) sequencer then sequentially floods the chip
with one nucleotide after another.
• If the next nucleotide that floods the chip is not a match, no voltage change will be recorded
and no base will be called.
Ion Torrent
Semiconductor based
technology
When a nucleotide is incorporated into a strand of DNA by a polymerase, a hydrogen ion is
released as a byproduct.
3rd Generation Sequencing Platforms
• Pacific Bioscience PacBio RS – Single molecule, real-time
analysis. SMRT DNA sequencing is performed on SMRT Cells,
each patterned with 150,000 zero mode waveguides or ZMWs.
Each ZMW contains a single DNA polymerase, providing the
window to observe DNA sequencing in real-time. The PacBio RS
system continuously monitors ZMWs in sets of 75,000 at a time.
• Oxford Nanopore – Nanopores offer a label-free, electrical,
single-molecule DNA sequencing method, obviating the need for
amplification or labeling, by detecting a direct electrical signal.
While the evolution of other technologies relies on improvements
in existing chemical, optical or bioinformatics procedures,
nanopores will bypass these to deliver a genuinely revolutionary
sequencing method.
• Exonuclease sequencing
Using a processive enzyme to cleave
individual nucleotides from a DNA strand
and pass them through a protein
nanopore. Oxford Nanopore has a
commercialisation agreement with Illumina
for this method.
• Strand sequencing
Identifying individual nucleotides on a
DNA strand as it passes intact through a
protein nanopore.
• Solid state sequencing
Using synthetic materials, rather than protein pores, to create nanopores.
http://vimeo.com/18630569
Nanopore sequencing technology development
Structure of the Protein Nanopore
Exonuclease sequencing: Combining a protein nanopore and processive enzyme for
the sequential identification of DNA bases as they pass through the pore.
Alpha haemolysin nanopore showing cyclodextrin adapter molecule (the DNA binding
site).
In the exonuclease sequencing method, a protein nanopore is coupled with a processive
enzyme, an exonuclease. The enzyme cleaves individual DNA bases from a DNA strand.
These bases enter the nanopore and undergo a binding event before passing through the
pore. During this binding event, they cause characteristic disruption in current that can be
used to identify the DNA bases in sequence. This method is combined with Oxford
Nanopore's proprietary electronics system for scaled-up nanopore sensing.
• Covalent attachment of a cyclodextrin molecule to
the inside surface of the nanopore.
• Acts as a binding site for individual DNA bases and
allows accurate measurement of their passage through
the nanopore binding site.
• Allows the identification of individual nucleoside 5’monophosphate molecules (DNA bases) to a standard
commensurate with a high resolution DNA sequencing
technology.
•
•
•
•
Electronic trace showing DNA bases in solution entering the nanopore.
The bases individually, transiently bind to the cyclodextrin adapter.
Each time a base passes through the pore there is a disruption in the current.
Diagram shows four different magnitudes of disruption which can be classified as C, G,
A or T.
Examples of Computational Solutions
• Cloud computing tools such as Crossbow is a cloud-computing
software tool that combines the aligner Bowtie and the SNP caller
SOAPsnp.
Executing in parallel using Hadoop, Crossbow analyzes data
comprising 38-fold coverage of the human genome in three hours using
a 320-CPU cluster rented from a cloud computing service for about
$85. Crossbow is available from http://bowtiebio.sourceforge.net/crossbow/ webcite.
• DNAnexus – a cost effective storage and computational toolset –
web based, running on the cloud
• Galaxy – suite of genomic tools developed and hosted by Penn
State. Web based and running on Penn servers with NHGRI
support
Development of Computational Biology
Programs
The data anaysis challenge is huge!
We have made significant progress and investment in the tools for genomic
studies
Progress is being made in building infrastructure for collection and
integration of clinical data
Focus needed on developing a program in computational biology to attract
faculty and research analysts
To assist clinicians and scientists with highthroughput genomic data analysis