Functional Genomics with Next-Generation Sequencing

1. Functional Genomics with Next-Generation Sequencing  Jen Taylor Bioinformatics Team CSIRO Plant Industry

2. CSIRO. INI Meeting July 2010 - Tutorial - Applications Capacity and Resolution Next generation sequencing Increasing capacity leads to increased resolution

3. CSIRO. INI Meeting July 2010 - Tutorial - Applications How a Genome Works? Parts Description Function? Interconnectedness? Comparisons Population - level Between genomes

4. CSIRO. INI Meeting July 2010 - Tutorial - Applications Application domains

5. CSIRO. INI Meeting July 2010 - Tutorial - Applications Impact of a Reference Genome

6. CSIRO. INI Meeting July 2010 - Tutorial - Applications Applications of Next Generation Sequencing Profiling of Variation Genetic variation Transcript variation Epigenetic variation Metagenomic variation Discovery Novel genomes Novel genes Novel transcripts Small / long non-coding RNA

7. CSIRO. INI Meeting July 2010 - Tutorial - Applications RNASeq Qualitative – transcript diversity Quantitative – transcript abundance Impact of NGS Observation of transcript complexity Transcript discovery Small / long non-coding RNA Analytical challenges Transcript complexity Compositional properties

8. CSIRO. INI Meeting July 2010 - Tutorial - Applications RNASeq

9. CSIRO. INI Meeting July 2010 - Tutorial - Applications RNASeq – Transcript Complexity

10. CSIRO. INI Meeting July 2010 - Tutorial - Applications RNASeq – Compositional properties Depth of Sequence Sequence count ˜ Transcript Abundance Majority of the data can be dominated by a small number of highly abundant transcripts Ability to observe transcripts of smaller abundance is dependent upon sequence depth

11. CSIRO. INI Meeting July 2010 - Tutorial - Applications RNASeq – Compositional properties Composition Sequence counts are a composition of a fixed number of total sequence reads Therefore they are sum-constrained and not independent Large variations in component numbers and sizes can produce artefacts

12. CSIRO. INI Meeting July 2010 - Tutorial - Applications RNASeq - Correspondence Good correspondence with : Expression Arrays Tiling Arrays qRT-PCR Range of up to 5 orders of magnitude Better detection of low abundance transcripts Greater power to detect Transcript sequence polymorphism Novel trans-splicing Paralogous genes Individual cell type expression

13. CSIRO. INI Meeting July 2010 - Tutorial - Applications Reference Genome - RNASeq

14. CSIRO. INI Meeting July 2010 - Tutorial - Applications Reference Genome - RNASeq

15. CSIRO. INI Meeting July 2010 - Tutorial - Applications Epigenome Protein-DNA interactions [ChIPSeq] Nucleosome positioning Histone modification Transcription factor interactions Methylation [MethylSeq] Impact of NextGen Whole genome profiling Resolution Analytical challenges Systematic bias Unambiguous mapping Robust event calling

16. CSIRO. INI Meeting July 2010 - Tutorial - Applications ChIPSeq

17. CSIRO. INI Meeting July 2010 - Tutorial - Applications ChIPSeq

18. CSIRO. INI Meeting July 2010 - Tutorial - Applications ChipSeq methods

19. CSIRO. INI Meeting July 2010 - Tutorial - Applications MethylSeq using Bisulfite conversion

20. CSIRO. INI Meeting July 2010 - Tutorial - Applications Limited publications from BS-Seq Mammals Methylation predominant occurs at CpG site Several publications in human One publications in mouse Plants Methylation occurs at CG, CHH, CHG sites Two publications in arabidopsis

21. CSIRO. INI Meeting July 2010 - Tutorial - Applications Problems of mapping BS-seq reads Reduced sequence complexity

22. CSIRO. INI Meeting July 2010 - Tutorial - Applications Problems of mapping BS-seq reads Increased search space

23. CSIRO. INI Meeting July 2010 - Tutorial - Applications ELAND Mapping reads to genome sequences Mapping reads to two converted genome sequences Cross match for reads mapping to multiple positions in converted genomes Mapping results were combined to generate methylation information Eland only allows 2 mismatches.

24. CSIRO. INI Meeting July 2010 - Tutorial - Applications BSMAP Based on HASH table seeding algorithm

25. CSIRO. INI Meeting July 2010 - Tutorial - Applications Re-mapping of Lister’s data using BSMAP

26. CSIRO. INI Meeting July 2010 - Tutorial - Applications Methylation pattern throughout chromosomes

27. CSIRO. INI Meeting July 2010 - Tutorial - Applications Partially / Unsequenced Genomes Options for dealing with partial or unsequenced genomes Wait for or generate the genome sequence ‘Borrow’ a reference genome from a phylogenetic neighbour Take a deep breath and ‘do denovo’ Denovo Genome Denovo Transcriptome

28. CSIRO. INI Meeting July 2010 - Tutorial - Applications Plant Genomes – Haploid Size

29. CSIRO. INI Meeting July 2010 - Tutorial - Applications Plant Genomes – Total Size

30. CSIRO. INI Meeting July 2010 - Tutorial - Applications Denovo RNA Seq Why transcriptome ? Large genome sizes with high repeat content are difficult to assemble Transcriptomes more constant size Enriched for functional content Aims : Transcript discovery Small /long non-coding RNA profiling Analytical challenges Assembly – ABySS, Velvet, Euler-SR Comparisons between non-discrete, overlapping transcripts Annotation Ploidy

31. CSIRO. INI Meeting July 2010 - Tutorial - Applications Summary – Impacts and Challenges RNASeq Increased resolution Increased power for transcript complexity and variation Analytical challenges – transcript complexity, compositional bias Large gains in small and long non-coding RNA profiling Epigenomics ChipSeq and MethylSeq Genome-wide with resolution Robust event calling is challenging Denovo transcriptomics Attractive option for large, repeat rich genomes

32. CSIRO. INI Meeting July 2010 - Tutorial - Applications Acknowledgements