1. Facilitator: Richard Bruskiewich
NGS Bioinformatics Workshop 1.1 Workshop Overview and Practical Informatics Considerations
March 7th, 2012
IRMACS, SFU
Facilitator: Richard Bruskiewich
Adjunct Professor, MBB
Welcome (title slide, 2 minutes)
Advanced thank you’s
Jim Mattson
Felix Breden
IRMACS team:
Pam Borghardt, IRMACS Centre, Managing Director
Brian Technical Director
Westgrid Team:
Ata Roudgar, Martin Siegert
Fiona Brinkman: for the kind permission to adapt a significant number of her introductory bioinformatics course MBB slides for portions of the workshop

2. Today’s Agenda – Part 1 Welcome and Acknowledgments
Some administrative details…
Introductions:
Facilitator
Participants
10 minute break
Introduce myself (5 minutes, 1 slide bio)
Administrative details of the workshop (5 minutes, 1 slide - admin details (schedule/rooms/dates/times/payment), material pre-requisites (computer)
Invite participants (round the table) to give ~1 minute talk (~30 minutes)
Your Name, department, lab, (your “port of origin”)
What is your research focus?
How can bioinformatics (NGS) support that research?
What NGS data of your own do you have to analyse *now*
Expectations for the workshop…
Survey Results (Part I)
Most of you have not yet taken a bioinformatics course; 1 – 2 of you took significant MBB courses
Overview of expectations from survey
10 minute break

3. Advance Acknowledgments
Jim Mattson: for championing the workshop idea
Felix Breden: for championing the idea of IRMACS bioinformatics support & endorsing this workshop
IRMACS team:
Pam Borghardt, IRMACS Managing Director: sponsorship
Brian Technical Director: workshop infrastructure
WestGrid Team:
Ata Roudgar, Martin Siegert: workshop HPC infrastructure
Fiona Brinkman: for her kind permission to adapt a number of her MBB introductory bioinformatics course slides for portions of the workshop

4. Lecture (12:30 – 14:30, Wednesdays) Demo/Lab (9:30 – 11:30, Thursdays)
Topic
Lecture (12:30 – 14:30, Wednesdays)
Demo/Lab (9:30 – 11:30, Thursdays)
Bioinformatics Overview (roughly equivalent to core MBB 441/741 topics)
Workshop Overview and
Practical Informatics Considerations
March 7th
March 8th
Sequence Formats, Databases and Visualization Tools
March 14th
March 15th
Sequence Alignment and Searching
March 21st
March 22nd
Principles of Structural Genomics and Overview of Next Generation Sequencing Technologies
March 28th
March 29th
Sequence Assembly Algorithms
April 4th
April 5th
Specific Applications
Sequence Assembly of Transcriptomes
May 2nd
May 3rd
Sequence Assembly of Whole Genomes
May 9th
May 10th
Annotation of de novo Assembled Sequences
May 16th
May 17th
Identification and Analysis of Sequence Variation
May 23rd
May 24th
Comparative Genomic Analysis and Visualization
May 30th
May 31st
Meta-Analysis of Newly Annotated Sequence Data
June 6th
June 7th

5. Venue
The workshop lectures and demo/labs will generally take place here, in the IRMACS Centre, Room (top floor, Applied Sciences Building) with the exception of the March 14th and May 9th lectures, plus the May 10th lab/demo for which there is a meeting conflict in IRMACS. These particular sessions will instead be convened in BioSci room B9242.
The lab/demo sessions on March 8th, 15th and 29th will end earlier, at 11 am, to accommodate the next scheduled event in IRMACS

6. Workshop Fee
Sign-up list to Barbara Sherman… will contact PI for billing(?)

7. NGS Bioinformatics Workshop 1
NGS Bioinformatics Workshop 1.1 Workshop Overview and Practical Informatics Considerations
Roadmap of the workshop (10 minutes, 3 slides - program revisited; + tech structure/flow diagram(?)
introductions

8. Facilitator Richard: A Brief Bio
Professional Experience
2009 – present, Adjunct Professor, MBB, SFU
, Research Scientist, Computational and Systems Biology, Bioinformatics, International Rice Research Institute (IRRI; irri.org)
, Postdoc, Human Analysis Team, Sanger Centre, Cambridge, UK
Academic Background
1999, PhD (Medical Genetics), UBC
1992, B.Sc. (Biochemistry, Molecular Biology& Genetics), UBC
1987, B.A. (Minor Computing), SFU
Personal
Originally from Edmonton; moved to GVRD in late teens and resided here for over 2 decades before travelling abroad to work
Wife is Filipina-Canadian (hence the job in the Philippines); 3 teenage kids (son in his late teens has just started in the SIAT program at SFU Surrey)
Returned last June to reside in Port Moody, at the foot of Burnaby Mountain

9. Participants “Around the table”
Your Name, department, lab, (PI)
(optional) Your “Port of Origin”
What is your research focus?
How can bioinformatics (NGS) support that research?
What NGS data of your own do you have to analyse *now*
Expectations for the workshop…
Introduce myself (5 minutes, 1 slide bio)
Administrative details of the workshop (5 minutes, 1 slide - admin details (schedule/rooms/dates/times/payment), material pre-requisites (computer)
Invite participants (round the table) to give ~1 minute talk (~30 minutes)
Your Name, department, lab, (your “port of origin”)
What is your research focus?
How can bioinformatics (NGS) support that research?
What NGS data of your own do you have to analyse *now*
Expectations for the workshop…
Survey Results (Part I)
Most of you have not yet taken a bioinformatics course; 1 – 2 of you took significant MBB courses
Overview of expectations from survey
10 minute break

10. 10 minute break…

11. Today’s Agenda – Part 2 What is Bioinformatics and why is it needed?
What is “Next Generation Sequencing”
Coping with the NGS bioinformatics challenge
The Workshop Road Map
Looking ahead…

12. What is bioinformatics?
NGS Bioinformatics Workshop 1.1 Workshop Overview and Practical Informatics Considerations
What is bioinformatics?

13. Bioinformatics is…
The development of computational methods for studying the structure, function, and evolution of genes, proteins, and whole genomes;
The development of methods for the management and analysis of biological information arising from genomics and high-throughput biological experiments.

14. Why is there Bioinformatics?
Fiona Brinkman Bioinfo Course
Summer 2002
Why is there Bioinformatics?
Huge datasets
Lots of new sequences being added
- Automated sequencers
Genome Projects
Metagenomics
- RNA sequencing, microarray studies, proteomics,…
Patterns in datasets that can be analyzed using computers

15. Need for informatics in biology: origins
Gramicidine S (Consden et al., 1947), partial insulin sequence (Sanger and Tuppy, 1951)
1961: tRNA fragments
Francis Crick, Sydney Brenner, and colleagues propose the existence of transfer RNA that uses a three base code and mediates in the synthesis of proteins (Crick et al., 1961) General nature of genetic code for proteins. Nature 192: In Microbiology: A Centenary Perspective, edited by Wolfgang K. Joklik, ASM Press. 1999, p.384
First codon assignment UUU/phe (Nirenberg and Matthaei, 1961)

16. Need for informatics in biology: origins
The key to the whole field of nucleic acid-based identification of microorganisms… …the introduction molecular systematics using proteins and nucleic acids by the American Nobel laureate Linus Pauling. Zuckerkandl, E., and L. Pauling. "Molecules as Documents of Evolutionary History." Journal of Theoretical Biology 8:
Another landmark: Nucleic acid sequencing (Sanger and Coulson, 1975)

17. Need for informatics in biology: origins
First genomes sequenced:
3.5 kb RNA bacteriophage MS (Fiers et al., 1976)
5.4 kb bacteriophage X (Sanger et al., 1977)
1.83 Mb First complete genome sequence of a free-living organism: Haemophilus influenzae KW20 (Fleischmann et al., 1995)
First multicellular organism to be sequenced: C. elegans (C. elegans sequencing consortium, 1998)
Early databases: Dayhoff, 1972; Erdmann, 1978
Early programs: restriction enzyme sites, promoters, etc… circa 1978.
1978 – 1993: Nucleic Acids Research published supplemental information

18. Genbank and associated resources doubles faster than Moore’s Law
Genbank and associated resources doubles faster than Moore’s Law! (< every 18 months)
(from the National Centre for Biotechnology Information)

19. Today: So many genomes…
As of mid-August 2010, according to the GOLD GenomesOnline database….
Eukaryotic genome projects are in progress? (Genome and ESTs) ( years ago)
Prokaryote genome projects are in progress?
5006 ( years ago)
Metagenome projects are in progress?
133 (Zero - 5 years ago)
TOTAL 6687 projects (As of Sept 2011: >10,000)

20. Information sources:
(Rhesus macaque) Robert F. Service. Science 311: (2006).
454 press release, May 31,
Wellcome Trust Sanger Institute press release, July 2,
Complete Genomics article in Bio-IT World:
Applied Biosystems press release, October 1,

25. The genome sequence is complete - almost!
The Human Genome
The genome sequence is complete - almost!
approximately 3.5 billion base pairs.

26. Work ongoing to locate all genes and regulatory regions and describe their functions… …bioinformatics plays a critical role

27. Identifying single nucleotide polymorphisms (SNPs) and other changes between individuals

28. Bioinformatics helps with……. Sequence Similarity Searching/Comparison
Fiona Brinkman Bioinfo Course
Summer 2002
Bioinformatics helps with……. Sequence Similarity Searching/Comparison
What is similar to my sequence?
Searching gets harder as the databases get bigger - and quality changes
Tools: BLAST and FASTA = early time saving heuristics (approximate methods)
Need better methods for SNP analysis!
Statistics + informed judgment of the biologist

29. Bioinformatics helps with……. Structure-Function Relationships
Fiona Brinkman Bioinfo Course
Summer 2002
Bioinformatics helps with……. Structure-Function Relationships
Can we predict the function of protein molecules from their sequence?
sequence > structure > function
Prediction of some simple 3-D structures possible (a-helix, b-sheet, membrane spanning, etc.)

30. Bioinformatics helps with……. Phylogenetics
Fiona Brinkman Bioinfo Course
Summer 2002
Bioinformatics helps with……. Phylogenetics
Can we define evolutionary relationships between organisms by comparing DNA sequences?
Lots of methods and software, what is the best analysis approach?

31. What is Next Generation Sequencing (ngs)?
NGS Bioinformatics Workshop 1.1 Workshop Overview and Practical Informatics Considerations
What is Next Generation Sequencing (ngs)?

32. Sanger (“dideoxy sequencing or chain termination”) Sequencing
Single stranded DNA from sample* extended by polymerase from primer then randomly terminated by dideoxy nucleotide (ddNTP)
Variable length DNA fragments radiolabelled or fluorescently detected ddNTP
*sample derived from amplified cDNA, genomic clones or whole genome shotgun

33. Sanger Pro’s & Con’s Advantages Disadvantage Relatively accurate
Relatively long (500 – 1500) bp reads
Disadvantage
Relatively costly in terms of reagents and relatively low throughput

34. Next Generation Sequencing (NGS)
Polonator
Roche 454
Sequence Assembly
on HPC
Life Tech. Ion Torrent
HeliScope
Illumina HiSeq
Life Tech SOLiD
Oxford Nanopore “GridION”
Pacific Biosciences SMRT Cell

35. (General) NGS Pro’s & Con’s
Advantages
Very high throughput
Very cheap data production
Disadvantages
Relatively short reads
Relatively higher error rates
Bioinformatics of assembly is much more challenging

36. General NGS Workflow Template preparation Sequencing & imaging
Genome alignment/assembly

37. Coping with the NGS bioinformatics challenge
NGS Bioinformatics Workshop 1.1 Workshop Overview and Practical Informatics Considerations
Coping with the NGS bioinformatics challenge

38. Challenge
Assembling “next generation sequence” (NGS) data requires a great deal of computing power and gigabytes memory
Software often can execute in parallel on all available computer processing unit (CPU) cores.
Many functional annotation processes (e.g. database searching, gene expression statistical analyses) also demand a lot of computing power

39. “High Performance Computing” and “Cloud Computing”
Computer Nodes
Network Storage
Your local workstation/ laptop

40. What is Cloud Computing?
Pooled resources: shared with many users (remotely accessed)
Virtualization: high utilization of hardware resources (no idling)
Elasticity: dynamic scaling without capital expenditure and time delay
Automation: build, deploy, configure, provision, and move without manual intervention
Metered billing: “pay-as-you-go, only for what you use
Cloud Computing

41. Cloud Bioinformatics Module
Input Job
Message Queue
Output Job
Message Queue
Task-
Specialized
Server
Start-up
(w/parameters)
Job Status
Notification
Customized
Machine
Image
Raw Data/
Results/
Snapshots

42. A More Complete Picture…
Web
Portal
Project
Relational
Database
Database
Loader
Raw Data + Results

43. Case Study in Bioinformatics on the Cloud
Used Amazon Web Services
Assembled ~99 raw NGS transcriptome sequence datasets from 83 species, on 16 Amazon EC2 instances with 8 CPU cores, 68 GB of RAM, ~200 hours of computer time, total run in less than one working day.
Each single machine of the required size would likely have cost at least ~$10,000 (and time) to purchase, and incur significant operating costs overhead (machine room space, power supplies, networking, air conditioning, staff salaries, etc.)
The above run could be started up in a few minutes and cost ~ $500 to complete. Once done, no machines left idling and unused…

44. Software for (NGS) Bioinformatics
Bundled with sequencing machines:
e.g. Newbler assembler with Roche 454
3rd party commercial:
DNA Star (
Geneious (
GeneWiz (
And others…
Open Source:
Lots (selected examples to be covered in this workshop)

45. What do I need to run bioinformatics software locally?
Some common bioinformatics software is platform independent, hence will run equally under Windows and UNIX (Linux, OSX)
Most other software targets Unix systems. If you are running Microsoft Windows and want to run such software locally, the easiest way to do this(?) is to install some version of Linux (suggest “Ubuntu”) as a dual boot or (less intrusively) as a guest operating system in a virtual machine, e.g.

46. But, what are *we* going to use here?

47. https://computecanada.org/
SFU / IRMACS
WestGrid is a consortium member of “Computer Canada”
“bugaboo” cluster: 4328 cores total: 1280 cores, 8 cores/node, 16 GB/node, x86_64, IB. Plus 3048 cores, 12 cores/node, 24GB/node, x86_64, IB. capability cluster, 40 Core Years
Access to other Westgrid resources through LAN and WAN
More details from Brian Corrie tomorrow…

48. Galaxy Genomics Workbench
(also

49. NGS Bioinformatics Workshop 1
NGS Bioinformatics Workshop 1.1 Workshop Overview and Practical Informatics Considerations
Roadmap of the workshop (10 minutes, 3 slides - program revisited; + tech structure/flow diagram(?)
The Workshop roadmap

50. Visualization of Sequence & Annotation
Road Map
What is Bioinformatics?
Visualization of Sequence & Annotation
NGS
Annotation
Sequence Assembly
Sequences
(Formats)
Sequence
Databases
Search &
Alignments

51. Specific Applications
Sequence Assembly of Transcriptomes
Sequence Assembly of Whole Genomes
Annotation of de novo Assembled Sequences
Identification and Analysis of Sequence Variation
Comparative Genomic Analysis and Visualization
Meta-Analysis of Annotated Sequence Data

52. Survey: Workshop Expectations I
How to find significance in the huge amount of data that Next Gen sequencing, but also microarrays etc. generate.
A basic understanding of how to analyse next generation sequencing data.
Learn some hands-on computer experience
learning to use software for analysing sequence data; what can be done and how to do it.
genome assembly + meta-analysis

53. Survey: Workshop Expectations II
The basics of alignment and SNP calling with next-gen sequencing, and what kind of programs are out there to do these tasks and then analyze the large datasets (I've been trying to figure this out on my own through reading the literature and it's quite time consuming so any info provided through the workshop would be very helpful - thanks)
The main workflow for processing sequence data from the beginning to the more specific paths of analyses. Also the concepts, significance of the adjustable parameters behind the various algorithms used in the workflow.

54. Survey: Workshop Expectations III
I expect to learn the basic bioinformatics tools.
Learn different sequence alignment software/technologies (i.e. BWA, Abyss, etc.). Learn more about the complexities of NGS sequencing
Next generation sequencing, data analysis etc.
Parameters regulating assembly of contigs. How to take raw data to an assembly, control the main parameters for assembly, mass analyze data for annotation and SNPs
How to compare expression profiles using RNA transcriptomes.
Want to learn new things

55. Survey: Operating System Being Used
Microsoft Windows on Intel/AMD – 14 (86.7%)
Most running Windows 7 (some XP & Vista)
One uses Linux through Westgrid and the IRMACS cluster
Some of you also thinking of running Linux
Apple OS X – 2 (13.3%)
Snow Leopard Release
Apple Lion, running Windows 7 using Parallels
Linux on Intel - 2 (13.3%)

56. Looking Ahead… What will you need for this workshop? Reading list:
Mainly, just a laptop running a web browser
(Optional) access to Linux/Unix locally (VM Player)
Reading list:
Will give review citations for future lectures
For next week, suggest that you surf to