vince buonaccorsi associate professor of biology juniata college
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NextGen sequencing for undergraduate research and curriculum. Vince Buonaccorsi Associate Professor of Biology Juniata College. NextGen Sequencing Technologies 454 Sequencing (Roche) SOLiD Sequencing (ABI). Nextgen sequencing: How it works. 454 GS FLX Titanium Series. - PowerPoint PPT PresentationTRANSCRIPT
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Vince BuonaccorsiAssociate Professor of BiologyJuniata College
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NextGen Sequencing Technologies
454 Sequencing (Roche)SOLiD Sequencing (ABI)
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Throughput400-600 million high-quality, filter-passed bases per run* 1 billion bases per day
Run Time 10 hours
Read LengthModal length = 500 bases, Average length = 400 bases
Data Trace data accepted by NCBI since 2005
http://www.454.com/products-solutions/system-features.asp
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http://www.454.com
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DNA Isolation:
1. Typical DNA extraction.2. Nebulization: mechanical DNA shearing.
http://www.roche-applied-science.com/publications/multimedia/genome_sequencer/flx_multimedia/wbt.htm
http://www.roche-applied-science.com/publications/multimedia/genome_sequencer/flx_multimedia/wbt.htm
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http://www.dnastar.com/media/flowgramlarge.jpg
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http://www.dnastar.com/media/flowgramlarge.jpg
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http://en.wikipedia.org/wiki/File:Phred_Score.svg
Q20 = -10 x log10(0.01)Q30 = -10 x log10(0.001)
Quality Scores
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Overview of Next Gen Sequencing
Medini et al. 2008. Microbiology in the post-genomic era. Nature Reviews Microbiology 6:419-430
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http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/index.htm
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http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/index.htm
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Summary of Capabilities
~ 10k per full run
454 ½ to 1 Million reads of 400bp ea = 200 to 400 Mb, 10hrs http://www.454.com/products-solutions/system-features.asp
SOLiD ½ Billion reads of 50 to 100bp ea= 25 to 50 Gb for 3 to 10 dayshttp://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/overviewofsolidsystem/index.htm
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http://www.nature.com/nmeth/journal/v6/n11s/pdf/nmeth.f.268.pdf
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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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http://www.openhelix.com/
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Metagenomic Analysis Of Windshield Splatter
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http://main.g2.bx.psu.edu/
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Pond et a. Genome Res. (2009) 19: 2144-2153Taylor et al. (2007) Current Protocols in Bioinformatics 10.5.1-10.5.25
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Soft Genetics: http://www.softgenetics.com/NextGENe.html
“The NextGENe Condensation Tool™, solves the 3 critical problems”
Reads too short High Error Rates Overwhelming Data Volume
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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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Symposium on Life Science Education, NC State, May 2009 http://statgen.ncsu.edu/slse
DOE adopt a genome, Cheryl Kerfeld http://www.jgi.doe.gov/education/genomeannotation.html
Bioinformatics Lab, Malcolm Campbell http://www.bio.davidson.edu/courses/Bio343/LabMethods_2009.
html
DNA Learning Center Gene Annotation labs, Bruce Nash http://www.dnalc.org/websites/dynamic.html
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http://www.jgi.doe.gov/education/adoptagenome/index.html
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http://www.jgi.doe.gov/education/assembly/teacher.html
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Application: de Novo Whole Genome Sequencingof Complex Genomes
•454 for deNovo sequencing of large genomes, connect over repeats
The GS FLX Titanium Series Paired End Protocol
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http://www.jgi.doe.gov/education/assembly/quality.html
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Apollo gene annotation of a genome region in Sebastes influenced by natural selection
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UCSC Genome Browser http://genome.ucsc.edu)
From Genes to Genomes in Non-Model Species
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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%)
http://www.juniata.edu/faculty/buonaccorsi/
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http://www.pacificbiosciences.com/
80,000 reads * 20,000 bp per read = 1.6GB for $100 in 10hrs start-finish
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SNP detection against reference
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Cofactor Genomics http://www.cofactorgenomics.com/home/about/cofactor-gives-back/
NSF REU: student internships http://www.nsf.gov/funding/pgm_summ.jsp?pims_id=5517&from=fund
NSF CCLI 200k-250k http://www.nsf.gov/pubs/2009/nsf09529/nsf09529.html 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) http://www.nsf.gov/funding/pgm_summ.jsp?pims_id=11691&org=BIO
Other: Innovative approach to a well-defined, important problem
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A guide to web resources
http://bib.oxfordjournals.org/cgi/reprint/8/1/6
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From 32000 ESTs Polymorphic Metabolic EST-SSRs
Download ESTs http://www.ncbi.nlm.nih.gov/nucest Find msats http://tandem.bu.edu/trf/trf.html Consolidate http://egassembler.hgc.jp/ http://toolkit.tuebingen.mpg.de/blastclu
st Identify using blastx http://blast.ncbi.nlm.nih.gov/Blast.cgi ID Metabolic process http://www.uniprot.org/help/uniprotkbs Insert introns http://genome.ucsc.edu Design primers http://probes.pw.usda.gov/batchprimer3/ Scan polymorphisms wet lab
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ApplicationsdeNovo Genomic SequencingDeep Sequencing for SNPsAncient DNARNA-SeqChip-SeqMetagenomics
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100-base reads and up to 40 Gb of data per sequencing run
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ApplicationsdeNovo Genomic SequencingDeep Sequencing for SNPsRNA-SeqChip-SeqMetagenomics
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https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=604416&tab=DetailInfo
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http://www.illumina.com/technology/sequencing_technology.ilmn
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454 Technology
Medini et al. 2008. Microbiology in the post-genomic era. Nature Reviews Microbiology 6:419-430
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http://www.hsls.pitt.edu/guides/genetics/obrc/
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Jonathan RothbergRothberg Institute of Childhood Diseases
454