Vince Buonaccorsi Associate Professor of Biology Juniata College

1. NextGen sequencing for undergraduate research and curriculum Vince Buonaccorsi Associate Professor of Biology Juniata College

2. NextGen Sequencing Technologies 454 Sequencing (Roche) SOLiD Sequencing (ABI) Nextgen sequencing: How it works

3. 454 GS FLX Titanium Series http://www.454.com/products-solutions/system-features.asp

4. Data from 454 sequencing http://www.454.com

5. http://www.roche-applied-science.com/publications/multimedia/genome_sequencer/flx_multimedia/wbt.htmhttp://www.roche-applied-science.com/publications/multimedia/genome_sequencer/flx_multimedia/wbt.htm DNA Isolation: • Typical DNA extraction. • Nebulization: mechanical DNA shearing. http://www.roche-applied-science.com/publications/multimedia/genome_sequencer/flx_multimedia/wbt.htm

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

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

20. Quality Scores Q20 = -10 x log10(0.01) Q30 = -10 x log10(0.001) http://en.wikipedia.org/wiki/File:Phred_Score.svg

21. Overview of Next Gen Sequencing Medini et al. 2008. Microbiology in the post-genomic era. Nature Reviews Microbiology 6:419-430

22. ABI Solid sequencing http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/OverviewofSOLiDSequencingChemistry/index.htm

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

25. 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 days http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/overviewofsolidsystem/index.htm

26. Dealing with the data http://www.nature.com/nmeth/journal/v6/n11s/pdf/nmeth.f.268.pdf

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) Dealing with the data

28. http://www.openhelix.com/

29. Metagenomic Analysis Of Windshield Splatter

30. Metagenomic analysis using galaxy http://main.g2.bx.psu.edu/

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

32. Galaxy workflow

34. Galaxy OUTPUT: TRIP A

35. 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 Dealing with the data

36. The Condensation tool

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. Soft genetics: NextGene

38. Chip-seq

39. Chip Seq: location of DNA-protein binding sites

41. 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 Into the Classroom

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

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

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

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

47. Apollo gene annotation of a genome region in Sebastes influenced by natural selection Genomics in student research

48. UCSC Genome Browser http://genome.ucsc.edu) 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%) • http://www.juniata.edu/faculty/buonaccorsi/ ADVANCED GENETICS

50. http://www.pacificbiosciences.com/ 80,000 reads * 20,000 bp per read = 1.6GB for $100 in 10hrs start-finish Third generation sequencing