The 454 and Ion PGM at the Genomics Core
Facility
Dr. Deborah Grove, Director for Genetic Analysis
Genomics Core Facility
Huck Institutes of the Life Sciences
Penn State University
Services
•DNA Sequencing
•Illumina, 454 and Ion PGM Next Gen Sequencing
•Microarray
•Genotyping – VNTRs, SNPs, Open Array
•qPCR by Real-Time
•DNA Synthesis
•DNA Extraction and Storage of DNA from Buccal Swabs
Sequencing at PSU Over the Years
Method
Manual
Gel
Bases per 1200
Day
Cycle Sequencing Reaction
Sequencing at PSU Over the Years
Method
Manual
Gel
Bases per 1200
Day
377 Gel
20,000
Sequencing at PSU Over the Years
Method
Manual
Gel
Bases per 1200?
Day
377 Gel
3100 –16
Capillary
20,000
100,000
Sequencing at PSU Over the Years
Method
Manual
Gel
Bases per 1200?
Day
377 Gel
3100 –16
Capillary
3730--96
Capillary
20,000
100,000
0.5 to 1
million
Sequencing at PSU Over the Years
Method
Manual
Gel
Bases per 1200?
Day
377 Gel
3100 –16
Capillary
3730--96
Capillary
SOLiD
Next-Gen
20,000
100,000
0.5 to 1
million
1 to 2
Billion
Next-Generation Sequencers:
Massively Parallel Platforms
• Roche 454FLX+ v2.8 – 500 million bases per run, 800 to 1000
bases
• Ion PGM 318 chip – 2 to 4 billion bases per run, 400 base length
• (Ion Proton)
Roche 454 – Next Generation Sequencer
• Pyrosequencing
• FLX+ v2.8 has 800 to
1000 bp read
• 160 million bases per
full slide
454 FLX +
454 Titanium Sequencing Applications
• Whole Genome – Shotgun
500 ngs dsDNA
• Whole Genome -- Paired-End (3kb, 8kb, 20kb)
15 to 30 ugs dsDNA
• Amplicon/Metagenomics 5 ngs
• Transcriptome RNA
DNA Fragmentation by nebulization
Fragment End Repair
AMPure Bead Clean up
Adaptor-Ligation
Small Fragment Removal
Library Quality Assessment and
Quantification
Primer Sets for Metagenomics
•16s Bacteria
•ITS for Fungus
•18s set for Fungus and other Eukaryotes, targeting
Protists
•Archaea targeting both Crenarchaeota and
Euryarchaeota
Amplicon Preparation
27F_M6CGTATCGCCTCCCTCGCGCCATCAGATATCGCGAGAGAGTTTGATCMTGGCTCAG
907R_M6TATGCGCCTTGCCAGCCCGCTCAGCCCCGTCAATTCMTTTGAGTTT
16S Variable Regions
27F_M6CGTATCGCCTCCCTCGCGCCATCAGATATCGCGAGAGAGTTTGATCMTGGCTCAG
907R_M6TATGCGCCTTGCCAGCCCGCTCAGCCCCGTCAATTCMTTTGAGTTT
Amplicons
27F
518R
907R
•One Bead
•One DNA library
•NTPs, Taq etc.
Ion PGM aka Ion Torrent
314 CHIP
316 CHIP
Approximate Cost from Genome Library thru
Sequencing
314 Chip
160 million bases, 400,000 reads
318 Chip
3 billion bases, 6 to 8 million reads
$1500 to $2000
Coverage
Full Plate = 1 million reads, 300 million bases
½ plate = 500,000 reads, 150 million bases
1 Quad = 200,000 reads, 60 million bases
Coverage
Full Plate = 1 million reads, 300 million bases
½ plate = 500,000 reads, 150 million bases
1 Quad = 200,000 reads, 60 million bases
http://www.youtube.com/embed/yVf2295JqUg
Coverage
Full Plate = 1 million reads, 300 million bases
½ plate = 500,000 reads, 150 million bases
1 Quad = 200,000 reads, 60 million bases
Full Plate = 1 million reads, 300 million bases
½ plate = 500,000 reads, 150 million bases
1 Quad = 200,000 reads, 60 million bases
Full Plate = 1 million reads, 300 million bases
½ plate = 500,000 reads, 150 million bases
1 Quad = 200,000 reads, 60 million bases
Applications
• Bacterial and Viral Genomes
•Amplicons
•Ampliseq Panels for SNP variants
Ampliseq Cancer Panel
•Only 10 ngs or less
•FFPE tissues
•Single Cells
•Libraries take 3.5 hours
•2800 hot spots
Ampliseq Custom Panels
Use Ion AmpliSeq Designer
P1 Chip 80 million reads, 10 to 15 billion bp
PII Chip 300 million reads, 500 to 800 billion bp
314 Chip 160 million bases, 400,000 reads
318 Chip
3 billion bases, 6 to 8 million reads
Pac Bio
•No amplification required
•Single molecule
•Several thousand base reads
(4 to 20 kb)
• Least GC-biased sequencing
•Run time 30 minutes
Applications
•Genomes: Finish Genomes and improve assembly with
extra long reads (4000bp average and up to 20,000)
• Genomic Complexity: Allow haplotype expansion, full
length transcripts and splice variants, repeat expansions,
minor variants
•Epigenome: Detects base modifications using kinetics
Thanks to:
The Huck Institutes of the Life Sciences
Lloyd and Dorothy Huck
And the others in the lab…