Transcript PowerPoint Presentation - The GS FLX Sequencer. What is it and

The GS FLX Sequencer. What is
it and what can we do with it?
Jo Stanton
Anatomy and Structural Biology
University of Otago
• The Equipment
• The Technology
• What can you do with the GS FLX?
• Multiple optical fibers are
fused to form an optical
array.
• Proprietary etching method
produces wells that serve as
picoliter reaction vessels.
• Each well is only able to
accept a single DNA bead.
• Reactions in the wells are
measured of the CCD
camera.
PicoTiterPlate device
DNA Library Preparation and Titration
4.5 h
and 10.5h
emPCR
Sequencing
8h
4.5h
 Well diameter: average of 44 µm
 200,000 reads obtained in parallel
 A single clonally amplified sstDNA bead is deposited per
well.
Amplified sstDNA library beads
Quality filtered bases
DNA Library Preparation and Titration
DNA capture bead
containing millions of
copies of a single clonal
fragment
Amplified sstDNA library beads
emPCR
Sequencing
 4 bases (TACG) cycled 42 times
 Chemiluminescent signal generation
 Signal processing to determine base
sequence and quality score
Quality filtered bases
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1. Raw data is processed
from a series of
individual images.
2. Each well’s data is
extracted, quantified,
and normalized.
3. Read data is converted
into flowgrams.
Metric and image viewing software
Signal output from a single well (flowgram)
Signal strength is determined by homopolymer length
4-mer
3-mer
2-mer
1-mer
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Flow Order
Sequence Specifications
• Generate over 100 million bases per 7.5 hour run.
• Achieve longer reads, averaging 200 - 300 bases.
• Attain higher throughput with over 400,000 reads
per run.
• Generate single-read accuracy that is greater than
99.5% over 200+ base pair reads.
• Benefit from consensus accuracy that is greater
than 99.99%.
*from Roche Genome Sequencer FLX System brochure.
Some Applications
Genome Sequencing
• Mammoth found in the permafrost near Lake
Taimyr, Russia (27,740±220 years before present).
• 0.73µg DNA extracted from 1g Mammoth
edentulous mandible.
• Made library and sequenced using GS20 system
• Results: 302,692 reads, average length 95bp.
28,000,000bp of sequence in total
• Sequences aligned to African elephant, human and
dog genomes.
• Mammoth was Female.
• Two Neanderthal genome projects. Using same
individual - 38,000 year old male bone from
Croatia. Genome size 3 billion base pairs.
• 454 sequencing has yielded 1 million base pairs to
date. Sanger/pyrosequencing yielded 65,000 base
pairs.
• Humans and Neanderthal diverged roughly
600,000 years ago. Arose from a population of
3000 individuals.
• <0.5% difference between Humans and
Neanderthal.
• Projects aim to finish genome sequencing in two
years.
• The hunt is on for more Neanderthals to sequence!
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Speciation
Comparative genomics
Process of domestication
Species relationships
Population genetics
Responses to climate change
Cause of extinctions
Other Genome Projects
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Pine
Barley
Campylobacter jejuni
Helicobacter pylori
Vibrio cholerae
Acinetobacter baumannii
Mycobacterium tuberculosis
Corynebacterium urealyticum
Myxococcus xanthus
Environmental Sequencing
• Characterization of Environments
• Deep mine microbial environments
• Tanzanian soil
• Sea water
• Bulk DNA prep from gut of ob/ob and +/+ litter mates.
• Combination of Sanger sequencing and GS20 Technology
to produce EGT’s (environmental gene tags)
• EGT breakdown: 94% bacterial, 3.6% eukaryotic (0.29%
mouse & 0.36% fungal), 1.5% archaeal, 0.61% viral.
• Increase in ratio of Firmicutes vs Bacteroidetes in obese
mice. Increase in Archaea in obese mice.
• Ob/ob microbiome is enriched for genes for initial
breakdown of dietary polysaccharides. Metabolic
pathways: starch/sucrose, galactose, butanoate.
• Transplantation of ob/ob or +/+ gut flora to germ free
mice. Ob/ob microbiome recipients gained significantly
more weight than +/+ recipients.
Genome Methylation
• Treat DNA sample with sodium bisulfite.
Deaminates unmethylated cytosine to uracil.
Methylated cytosine is unchanged.
• Amplify regions of interest using PCR
• Sequence and compare to untreated sample.
• Example shows methylation of the CpG
island in the p16 promoter from two cell
lines.
Mutation Detection/Population
Genetics
• PCR amplify defined regions of genome from
individuals.
• Sequence and compare.
• Ultra Deep Sequencing
• Example: EGFr gene mutations in Cancer patient.
Ultra-Deep Sequencing of EGFR
from Lung Carcinoma Patients
Matthew Meyerson, Dana Farber Institute and 454 Life Sciences
A total of 11 PCR amplicons, ranging in size between 85 and 156 base pairs, were generated to cover
Amplicons
Exon 22
Exon 21
Exon 20
Exon 19
Exon 18
Coverage
exons 18-22 of EGFR. Each target region was individually amplified and quantified. Before emulsionbased PCR amplification and sequencing all amplicons were pooled in equimolar ratios
Sequencing By Synthesis
Biopsy
PCR
Instant cloning:
Seperation
of single molecules
during emPCR
454 =
Single Molecule
Sequencing
Comparison of
single sequences
emPCR
Sequencing
Identification of
mutations
• A lung adenocarcinoma
sample previously
shown by Sanger
sequencing to contain
mutation G719S was
sequenced on the GS20
platform
• Two mutations easily
seen at 16 – 18% with
800 fold coverage
• Only one is seen in the
Sanger sequence
 Following 12.5 months of
erlotinib treatment patient
12.3 relapsed with a massive
pleural effusion. Pathological
examination of a fibrin clot
from the pleural effusion
fluid showed very low tumor
content in the isolated
sample.
• Del-4 mutation at 3%
abundance (Exon 19)
• In addition, T790M
mutation, at 2% (Exon 20)
shown in previous studies to
confer resistance to TKI
inhibitors
Matthew Meyerson, Dana Farber Institute and 454 Life Sciences
Transcriptome analysis
• Isolate RNA from cell/tissues and convert to
cDNA. Sequence.
• GIS-PET - 462,626 in 1 run. 1476 novel genes
identified (PET=paired end ditagging)
• SAGE > 800,000
• EST approach
• Example: Arabidopsis transcriptome
Aribidopsis transcriptome from 8
day old seedlings
• 541,852 ESTs
• 17,449 gene loci. Close to complete transcriptome
coverage.
• small, medium and long transcripts detected
equally.
• No sequencing bias to either 3’ or 5’ ends of
transcripts.
• ESTs not contaminated by genomic DNA intron/exon boundaries clearly preserved
• 16,698 ESTs not in dbEST.
• Many of these are the first evidence that predicted
genes are transcribed.
• 60 previously unidentified loci shown as possible
protein-coding sequences.
• Traditional sequencing relies on bacterial cloning.
Therefore cloned products toxic to E.coli will not
be detected. Not a problem for 454 technology.
• 454 chemistry is not hampered by trasitionally
difficult to clone sequence.
• Gene expression profiling possible using this
approach. Digital Northerns and an open system.
• Ideal for non-model systems
Micro RNA Analysis
sequence.otago.ac.nz
• GS FLX Brochure
• Publications list
• First point of call for information regarding
the Anatomy GS FLX facility.