Vince BuonaccorsiAssociate Professor of BiologyJuniata College

NextGen Sequencing Technologies

454 Sequencing (Roche)SOLiD Sequencing (ABI)

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

http://www.454.com

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

http://www.dnastar.com/media/flowgramlarge.jpg

http://www.dnastar.com/media/flowgramlarge.jpg

http://en.wikipedia.org/wiki/File:Phred_Score.svg

Q20 = -10 x log10(0.01)Q30 = -10 x log10(0.001)

Quality Scores

Overview of Next Gen Sequencing

Medini et al. 2008. Microbiology in the post-genomic era. Nature Reviews Microbiology 6:419-430

http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/index.htm

http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/index.htm

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

http://www.nature.com/nmeth/journal/v6/n11s/pdf/nmeth.f.268.pdf

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)

http://www.openhelix.com/

Metagenomic Analysis Of Windshield Splatter

http://main.g2.bx.psu.edu/

Pond et a. Genome Res. (2009) 19: 2144-2153Taylor et al. (2007) Current Protocols in Bioinformatics 10.5.1-10.5.25

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

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.

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

http://www.jgi.doe.gov/education/adoptagenome/index.html

http://www.jgi.doe.gov/education/assembly/teacher.html

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

http://www.jgi.doe.gov/education/assembly/quality.html

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

 UCSC Genome Browser http://genome.ucsc.edu)

From Genes to Genomes in Non-Model Species

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/

http://www.pacificbiosciences.com/

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

SNP detection against reference

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

A guide to web resources

http://bib.oxfordjournals.org/cgi/reprint/8/1/6

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

ApplicationsdeNovo Genomic SequencingDeep Sequencing for SNPsAncient DNARNA-SeqChip-SeqMetagenomics

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

ApplicationsdeNovo Genomic SequencingDeep Sequencing for SNPsRNA-SeqChip-SeqMetagenomics

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

http://www.illumina.com/technology/sequencing_technology.ilmn

454 Technology

Medini et al. 2008. Microbiology in the post-genomic era. Nature Reviews Microbiology 6:419-430

http://www.hsls.pitt.edu/guides/genetics/obrc/

Jonathan RothbergRothberg Institute of Childhood Diseases

454