The 454 and Ion PGM at the Genomics Core Facility

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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
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• 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

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Sequencing at PSU Over the Years
Method Manual Gel
Bases per Day 1200
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Cycle Sequencing Reaction
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Sequencing at PSU Over the Years
Method Manual Gel 377 Gel
Bases per Day 1200 20,000
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Sequencing at PSU Over the Years
Method Manual Gel 377 Gel 3100 16 Capillary
Bases per Day 1200? 20,000 100,000
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Sequencing at PSU Over the Years
Method Manual Gel 377 Gel 3100 16 Capillary 3730--96 Capillary
Bases per Day 1200? 20,000 100,000 0.5 to 1 million
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Sequencing at PSU Over the Years
Method Manual Gel 377 Gel 3100 16 Capillary 3730--96 Capillary SOLiD Next-Gen
Bases per Day 1200? 20,000 100,000 0.5 to 1 million 1 to 2 Billion
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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)

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Roche 454 Next Generation Sequencer

• Pyrosequencing
• FLX v2.8 has 800 to 1000 bp read
• 160 million bases per
• full slide

454 FLX
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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

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DNA Fragmentation by nebulization
Fragment End Repair
AMPure Bead Clean up
Adaptor-Ligation
Small Fragment Removal
Library Quality Assessment and Quantification
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Primer Sets for Metagenomics

• 16s Bacteria
• ITS for Fungus
• 18s set for Fungus and other Eukaryotes,
  targeting Protists
• Archaea targeting both Crenarchaeota and
  Euryarchaeota

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Amplicon Preparation
27F_M6CGTATCGCCTCCCTCGCGCCATCAGATATCGCGAGAGAGTTTGA
TCMTGGCTCAG 907R_M6TATGCGCCTTGCCAGCCCGCTCAGCCCCG
TCAATTCMTTTGAGTTT
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16S Variable Regions
27F_M6CGTATCGCCTCCCTCGCGCCATCAGATATCGCGAGAGAGTTTGA
TCMTGGCTCAG 907R_M6TATGCGCCTTGCCAGCCCGCTCAGCCCCG
TCAATTCMTTTGAGTTT
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Amplicons
27F 518R 907R
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• One Bead
• One DNA library
• NTPs, Taq etc.

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Ion PGM aka Ion Torrent
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314 CHIP
316 CHIP
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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
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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

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Coverage
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• 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
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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

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• Full Plate 1 million reads, 300 million bases
• ½ plate 500,000 reads, 150 million bases
• 1 Quad 200,000 reads, 60 million bases

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• Full Plate 1 million reads, 300 million bases
• ½ plate 500,000 reads, 150 million bases
• 1 Quad 200,000 reads, 60 million bases

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• Applications
• Bacterial and Viral Genomes
• Amplicons
• Ampliseq Panels for SNP variants

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• Ampliseq Cancer Panel
• Only 10 ngs or less
• FFPE tissues
• Single Cells
• Libraries take 3.5 hours
• 2800 hot spots

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Ampliseq Custom Panels Use Ion AmpliSeq
Designer
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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
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Pac Bio

• No amplification required
• Single molecule
• Several thousand base reads
• (4 to 20 kb)
• Least GC-biased sequencing
• Run time 30 minutes

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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

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Thanks to The Huck Institutes of the Life
Sciences Lloyd and Dorothy Huck And the others
in the lab