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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. Services DNA Sequencing Illumina, 454 and Ion PGM Next Gen Sequencing Microarray
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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
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
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.
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
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…