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GS Junior System – First Results
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IMPORTANT NOTICEIntended Use
Unless explicitly stated otherwise, all Roche Applied Science and 454 Life Sciences products and services
referenced in this presentation / document are intended for the following use:
For Life Science Research Only. Not for Use in Diagnostic Procedures.
www.454.com
Hemorrhagic Fever Virus Discovery in Native Host
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http://www.ncbi.nlm.nih.gov/pubmed/21544192
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Hemorrhagic Fever Virus Discovery in Native Host• Darted Red Colobus monkey in the wild in Kibale National Park, Uganda• Collected blood sample, isolated viral RNA/DNA• Sequenced on GS Junior System• Assembled using CLC genomics assembler, screened out host contigs• Identified two novel SHFV (simian hemorrhagic fever virus) strains• Generated near full-length viral sequences by filling in short gaps with
PCR/Sanger sequencing and 3’RACE• Significant findings:
– Not one, but TWO divergent SHFV viruses were present in one individual– Red Colobus monkey is a native reservoir for these pathogenic viruses– DNA was isolated from a healthy animal, demonstrating that these viruses
can hide in apparently healthy individuals – Consequences for human contact, spreading viruses through research
colonies
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Plant Pathogen Sequencing
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http://www.ncbi.nlm.nih.gov/pubmed/21131493
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Plant Pathogen Sequencing
• Erwina amylovora, fire blight pathogen, isolated from blackberry in Illinois
• Commercial apple and pear blight, reported in 1790s• 3.81 Mb genome, 53% GC, three circular plasmids• Sequenced using 3/8 of GS FLX run and one GS Junior run (equal to
four GS Junior runs)• 31x coverage, 375 bp avg. read length • Assembled by 454 GS De Novo Assembler into 29 contigs, gaps
closed in silico using LaserGene• Used GenDB to assign gene function for 3869 coding sequences• Comparative genomics with related strains
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Rare Variant Detection for HIV-1Saliou et al. Antimicrob. Agents Chemother April 2011
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Why Detect HIV Variants?
• HIV variants or “quasispecies” can use CCR5 and/or CXCR4 cell-surface receptors to enter cells
• Drugs that block CCR5 receptors work only if CXCR4-binding variants are absent
• As a result, there are tests to be sure that there are no CXCR4 binding viral variants before administering this class of HIV drugs to an individual
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Why use 454 Sequencing System?Potential to deliver speed, ease of use, cost savings• Current high sensitivity assays can detect viral variants at 0.3%, but
are slow, expensive and difficult• Current Sanger sequencing assays are rapid, cheap but cannot detect
quasi-species below 10-20%• Sensitivity at 0.3% can best predict treatment outcomes• 454 Sequencing Systems can deliver sequencing specificity for ~25
samples in one GS Junior run
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Experimental Design
• 415 base cDNA amplicon covering V3 env. region of HIV-1• Nested RT-PCR to generate amplicons with MIDs• 23 individual samples obtained ~3,500 reads/sample, sequenced
in one GS Junior run• GS AVA software used to align to reference• Processed the reads using third party prediction software• Detected quasispecies to 0.6% reliably• Calculated mean error rate of .000853 for pyrosequencing from
control plasmids!
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Results
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Summary- 84,000 reads- 23 samples- 0.6% detection limit
Critical Factors- 415 bp amplicon- 1600 or more reads per sample
Detection limited by software that predicts phenotype
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First Publication using GS Junior System Data
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Summary of Results
• Sequencing of MHC class I transcripts in macaques to discover all expressed transcripts from common class I haplotypes
• Sequenced 3 amplicons from ~440 to 620 bases• Combination experiment
– 7 individuals on GS FLX System, 3 using GS Junior System– Identified all sequences found previously – Discovered 2x more haplotypes than with previous Sanger-based
approach• 440-600 base amplicons allow resolution of haplotypes that are
impossible with 190 base amplicons
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GS Junior SystemPrimary applications
• de novo sequencing– sequencing of whole microbial, viral and other small genomes
• Targeted sequencing– Using sequence capture, PCR, amplicons, transcriptome cDNA
sequencing– Genotyping, rare variant detection, somatic mutation detection,
disease associated genes, genomic regions• Metagenomics
– characterization of complex environmental samples (16s rRNA and shotgun)
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Whole Genome Shotgun SequencingSequencing of three representative bacterial genomes
System GS FLX
GS Junio
r GS FLXGS Junior GS FLX
GS Junior
Organism E. coli K-12 T. thermophilus C. jejuniGenome Size(in Kb) 4563 2120 1600Avg. ContigSize (in Kb) 39 58 44 53 49 46N50
ContigSize(in Kb) 84 112 112 121 115 95Largest
ContigSize (in Kb) 209 352 474 578 304 173Number OfContigs 115 78 48 40 33 35
de novo Assemblies at 25x coverage using GS Junior and GS FLX Titanium reads
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Data from GS Junior System Shotgun RunsVariety of different microbes, early access site dataRun
Passed Filter Reads Avg Length Total Bases
1 117,636 445.1 52,350,2542 83,045 323.6 26,867,0863 90,415 386.7 34,954,1014 128,225 350.6 44,939,6535 43,321 353.2 15,297,8286 66,100 367.2 24,265,4077 100,335 433.4 43,475,7248 79,145 394.8 31,242,8759 109,894 422.6 46,430,503
10 108,779 437.8 47,613,70811 94,605 457.4 43,271,23312 61,975 398.7 24,706,55713 99,273 384.2 38,134,16514 115,776 429.5 49,716,84915 115,972 419.3 48,622,87416 115,031 414.4 47,661,170
Average 95,595 401 38,721,874
3kb paired end- 1M base genome, 1 run, one scaffold
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Read Length
• One GS Junior System run produces reads from 50-600 or more in length
• Average is in 330-400 base range• Most reads are in the 450-550 base range
Num
ber o
f re
ads
Readlength (bases)
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CFTR Exon Resequencing on GS Junior System
Experimental design:• 11 Coriell samples with known
mutations in CF gene• Each sample was MID-labeled (11
MIDs)• Amplified all 27 coding exons with
34 amplicons • Mixed 11x34 = 374 amplicons• Sequenced in 1 GS Junior System
run• Average coverage 182x• 96% of the reads mapped back to
the CF gene region
Numbers of reads per amplicon(across 11 samples)
0
100
200
300
400
500
600
0 50 100 150 200 250 300 350 400
374 Individual Amplicons#
of R
eads
Coverage graph: range 27-551xSince multiplex PCR reactions could not be normalized, PCR efficiency dictated the coverage levels for each amplicon
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Sizes of actual amplicons
CFTR Variant Detection by GS Junior System
• AVA output – showing 5 of 11 samples vs. variants discovered
ΔF508: known, phenotype-associated CFTR mutation
Heterozygous
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GS Junior and GS FLX reads are equivalent CFTR Variant Detection ΔF508
R668C
known, phenotype-associated CFTR mutationSynonymoussame mutation detected in two separate, overlapping, amplicons
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GS Junior Haplotyping of HLA Loci• Read length and clonality critical for resolution of individual
haplotypes- sequencing covers multiple alleles in each clonal read!• The longer the read, the better haplotype discrimination-
– below 200 bases=very poor– 200-300=poor– 300-500=good– 500-800=excellent
Allele 1
Allele 2
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Studying SIV using GS Junior System
• Ben Burwitz in Dave O’Connor’s lab, Univ. of Wisconsin• Follow changes in GAG gene as virus evolves to evade immune
response• Find genome-wide mutations in viral pool
Simian Immunodeficiency Virus
Rhesusmacaque
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Amplicon Sequencing- Basic Amplicon454 amplicon design using tailed primers
454 Titanium B-primer (21 bp)
MID
MID
key
keyA
B
454 Titanium A-primer (21 bp)
Sequence of interest
Locus-specific PCR amplification
200-600 bp
emPCR Amplification and sequencing
• Long reads required to sequence through the locus specific primer, enable haplotyping over longer distances
• 100s to 1000s of amplicon clones sequenced simultaneously
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Amplicon Sequencing- Long Range AmpliconsUsing long range amplicons for whole viral or other genomic region sequencing
Sequence of interest
Locus-specific long range PCR amplification
1,500-15,000 or more bp
emPCR Amplification and sequencing
MIDkey
A BMID
key454 Titanium A-primer (21 bp)
454 Titanium B-primer (21 bp)
Ligate sheared amplicon into 454 primers using gDNA protocol
Shear to 400-600 bases using gDNA protocol
BA
BABA
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SIV Genome Sequencing
SIV Genome(Viral RNA) 0bp 10535bp
Direct Amplicon
SIV Proteome
Full Genome
* Slide courtesy of U Wisconsin
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SIV Genome Sequencing – Direct Amplicon
Read Length (bp)
Num
ber o
f Rea
ds
354bp
# of Samples - 28
Total Reads - 82,079
Median Length - 356bp
* Slide courtesy of U Wisconsin
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Viral Mutations in the Structural SIV Protein Gag evolve to escape immune response
Mutations in the SIV protein Gag affect viral fitness- Gag protein is the ‘particle making machine’
* Slide courtesy of U Wisconsin
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Viral Mutations in the Structural SIV Protein Gag evolve to escape immune response
Mutations in the SIV protein Gag affect viral fitness- Gag protein is the ‘particle making machine’
* Slide courtesy of U Wisconsin
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SIV Genome Sequencing
SIV Genome(Viral RNA) 0bp 10535bp
Direct Amplicon
SIV Proteome
Full Genome
* Slide courtesy of U Wisconsin
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SIV Genome Sequencing - Amplicons
Read Length (bp)
Num
ber o
f Rea
ds
Total Reads - 59,097
Median Length - 321bp
~2kb~2kb
~2kb~2kb
* Slide courtesy of U Wisconsin
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SIV Full Genome Sequencing Coverage
SIV Genome - Base Pair Position
Num
ber o
f Rea
ds
* Slide courtesy of U Wisconsin
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454 Sequencing System vs. Sanger
Animal 1 Animal 2 Animal 3
* Slide courtesy of U Wisconsin
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Ben’s Conclusions
•GS Junior System detects low frequency genetic variants that are missed by traditional Sanger sequencing
•A bench-top GS Junior System improves turn around time and can be readily adapted to small academic lab settings
Acknowledgements
Ben BurwitzRoger WisemanShelby O’Connor
Dawn DudleyJulie Karl
Simon LankCharlie BurnsEricka BeckerBen Bimber
Dave O’Connor
O’Connor LabWatkins LabJonah Sacha
Matt ReynoldsNick ManessNancy WilsonDavid Watkins
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Inherited Disease• Looking for rare mutations in affected individuals• Target gene from GWAS study• Two PCR approaches- long range PCR and short amplicon• MID sequences used to distinguish individuals in a pool
Target Gene1 2 3 4 5 6 7 8 9 10 11 12 13 14
MID 1
MID 2
MID 3
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Long Range Amplicon Sequencing Results
Run Reads
Average Read Length (bases)
Total Bases
# of Sample Sequenced *
1 96,947 385 37,363,295 8
2 134,252 389 52,263,214 9
3 149,809 417 62,540,439 10
4 143,498 417 59,930,800 10
5 151,370 394 59,732,290 8
Shotgun processing
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Small Amplicon Sequencing ResultsAmplicon Processing
Run Reads Average Read Length (bases) Total Bases # of Sample
Sequenced
1 72,191 322 23,289,440 11
2 75,424 313 23,664,312 12
3 84,441 325 27,443,160 12
4 101,395 339 34,394,604 12
5 60,243 435 26,248,268 12
6 25,884 374 9,690,154 12
7 70,406 424 29,905,454 12
8 71,587 434 31,064,908 11
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Amplicon Coverage- Accurate Pooling Required!
Indi
vidu
al
Sam
ples
Amplicons
Poor performing SamplePoor Performing AmpliconSampling Variability
Poorly Pooled Amplicon
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Sample ID ASP Result GS Junior Agreeme
nt
1 Heterozygous
50.94% / 106 Y
2 Heterozygous
52.5% / 200 Y
3 Heterozygous
39.33% / 178 Y
4 Homozygous 94% / 100 Y
5 Heterozygous 48% / 125 Y
6 Heterozygous
47.06% / 221 Y
7 Homozygous
99.18% / 243 Y
8 Heterozygous
46.71% / 167 Y
9 Heterozygous
46.07% / 191 Y
10 Heterozygous
54.17% / 24 Y
11 Homozygous
97.57% / 288 Y
12 Heterozygous
42.33% / 163 Y
13 Heterozygous
41.88% / 191 Y
14 Heterozygous
47.02% / 151 Y
15 Heterozygous
48.07% / 441 Y
16 Heterozygous
17.86% / 252 N
17 Heterozygous
50.32% / 157 Y
18 Heterozygous
16.18% / 272 Y
19 Heterozygous
14.85% 330 Y
Allele-Specific PCR:Selective PCR amplification of one of the alleles to detect Single Nucleotide Polymorphism (SNP).
Selective amplification is usually achieved by designing a primer such that the primer will match/mismatch one of the alleles at the 3'-end of the primer.
Wild-Type Primer Set Assay Primer Set Genotype
Sample 1 Amplified Not Amplified Wild Type
Sample 2 Amplified Amplified Heterozygous
Sample 3 Not Amplified Amplified Homozygous
Verification of Novel Mutations
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Pathogen Discovery on the GS Junior System•Case from Sandton, South Africa•Infected paramedic during transfer, nurse at
hospital, cleaning staff, and nurse of paramedic- 4/5 did not survive
Serum and tissue samples from victims were subjected to unbiased pyrosequencing, yielding within 72 hours of sample receipt, multiple discrete sequence fragments that represented approximately 50% of a prototypic arenavirus genome.
•Recapitulated GS FLX System study in single GS Junior System run
•250 Hits to LuJo Virus covering 57% of the L-segment and 79% of the S-segment
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Coming Soon
• GS Junior System Publications in – Metagenomic characterization of human environments– Whole Genome Sequencing of bacterial pathogens– Rare variant discovery in human disease- GWAS follow up
experiments– Viral pathogen sequencing– Many more!
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GS Junior System First ResultsDisclaimer & Trademarks
Disclaimer:For life science research only. Not for use in diagnostic procedures. Trademarks:454, 454 LIFE SCIENCES, 454 SEQUENCING, EMPCR, GS FLX, GS FLX TITANIUM, GS JUNIOR and SEQCAP are trademarks of Roche.Other brands or product names are trademarks of their respective holders.
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