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Vince Buonaccorsi Associate Professor of Biology Juniata College.

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Presentation on theme: "Vince Buonaccorsi Associate Professor of Biology Juniata College."— Presentation transcript:

1 Vince Buonaccorsi Associate Professor of Biology Juniata College

2 NextGen Sequencing Technologies 454 Sequencing (Roche) SOLiD Sequencing (ABI)

3 Throughput million high-quality, filter-passed bases per run* 1 billion bases per day Run Time10 hours Read Length Modal length = 500 bases, Average length = 400 bases DataTrace data accepted by NCBI since 2005

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5 DNA Isolation: 1.Typical DNA extraction. 2.Nebulization: mechanical DNA shearing. science.com/publications/multimedia/genome_sequencer/flx_multimedia/wbt.htm

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20 Q20 = -10 x log 10 (0.01) Q30 = -10 x log 10 (0.001) Quality Scores

21 Overview of Next Gen Sequencing Medini et al Microbiology in the post-genomic era. Nature Reviews Microbiology 6:

22 mSequencing/OverviewofSOLiDSequencingChemistry/index.htm

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25 Summary of Capabilities ~ 10k per full run 454½ to 1 Million reads of 400bp ea = 200 to 400 Mb, 10hrs SOLiD½ Billion reads of 50 to 100bp ea= 25 to 50 Gb for 3 to 10 days

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27  Workshops/Tutorials to Learn Freeware  Metagenomic analysis using Galaxy  Vender Assisted Analysis  CHIP-seq analysis using Soft Genetics  Miscellaneous Web Programs/ListServes  SEQanswers (http://seqanswers.com)

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29 Metagenomic Analysis Of Windshield Splatter

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31 Pond et a. Genome Res. (2009) 19: Taylor et al. (2007) Current Protocols in Bioinformatics

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35  Soft Genetics: Soft Genetics:  “The NextGENe Condensation Tool™, solves the 3 critical problems”  Reads too short  High Error Rates  Overwhelming Data Volume

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37  For many applications, the data can be analyzed in four major steps:  1. Convert sample file format.  2. Condense sequence reads (polish data)  Low frequency instrument errors  Elongating the read lengths  Optional reduction of number of reads to increase analysis speed  3. Align or assemble reads.  4. View and export results.

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41  Symposium on Life Science Education, NC State, May 2009   DOE adopt a genome, Cheryl Kerfeld   Bioinformatics Lab, Malcolm Campbell   DNA Learning Center Gene Annotation labs, Bruce Nash 

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44 Application: de Novo Whole Genome Sequencing of Complex Genomes 454 for deNovo sequencing of large genomes, connect over repeats The GS FLX Titanium Series Paired End Protocol

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47 Apollo gene annotation of a genome region in Sebastes influenced by natural selection

48 UCSC Genome Browser From Genes to Genomes in Non-Model Species

49  Learning Objectives  Understand capabilities of emerging tools used in genetic analysis  Verbal fluency in basic principles and practices of genetics  Understand how geneticists study evolution at a variety of time scales  Independent mastery and creative thought in one area of genetics via writing research proposal  Assessment  Demonstrate understanding of material through two midterms (30%)  Presentations, case studies (25%)  Write a research proposal (45%) 

50   80,000 reads * 20,000 bp per read = 1.6GB for $100 in 10hrs start-finish

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52 SNP detection against reference

53  Cofactor Genomics   NSF REU: student internships   NSF CCLI 200k-250k   Projects that:  Build on the current understanding of how people learn  Have the potential to transform undergraduate STEM education  Research Coordination Networks in Biological Sciences (RCN)   Other: Innovative approach to a well-defined, important problem

54  A guide to web resources

55  From ESTs  Polymorphic Metabolic EST-SSRs  Download ESTs  Find msats  Consolidate   Identify using blastx  ID Metabolic process  Insert introns  Design primers  Scan polymorphisms wet lab

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60 Applications deNovo Genomic Sequencing Deep Sequencing for SNPs Ancient DNA RNA-Seq Chip-Seq Metagenomics

61 100- base reads and up to 40 Gb of data per sequencing run

62 Applications deNovo Genomic Sequencing Deep Sequencing for SNPs RNA-Seq Chip-Seq Metagenomics

63 https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=604416&tab=DetailInfo

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69 454 Technology Medini et al Microbiology in the post-genomic era. Nature Reviews Microbiology 6:

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76 Jonathan Rothberg Rothberg Institute of Childhood Diseases 454


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