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Large-scale genome projects

Strategy. Libraries. Sequencing. Assembly. Closure. Annotation. Release. Large-scale genome projects. Sequencing DNA molecules in the Mb size range All strategies employ the same underlying principles: Random Shotgun sequencing. Genomic DNA. Shearing/Sonication.

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Large-scale genome projects

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  1. Strategy Libraries Sequencing Assembly Closure Annotation Release Large-scale genome projects • Sequencing DNA molecules in the Mb size range • All strategies employ the same underlying principles: • Random Shotgun sequencing

  2. Genomic DNA Shearing/Sonication Subclone and Sequence Shotgun reads Assembly Contigs Finishing read Finishing Complete sequence

  3. Nucleotide Database Growth

  4. EMBL breakdown by organism

  5. EMBL Release 65

  6. Progress on Large Sequencing Projects

  7. Strategy Libraries Sequencing Assembly Closure Annotation Release Strategies for sequencing • How big can you go?? • Large-insert clones • cosmids 30-40 kb • BACs/PACs 50 - 100 kb • Whole chromosomes • Whole genomes

  8. Genome size and sequencing strategies Genome size (log Mb) 4 0 1 2 3 H.sapiens (3000 Mb) D.melanogaster (170 Mb) C.elegans (100Mb) P.falciparum (30 Mb) S.cerevisiae (14 Mb) E.coli (4 Mb) Whole genome shotgun (WGS) Clone-by-clone Whole Chromosome Shotgun (WCS) Whole Genome Shotgun (WGS) with Clone ‘skims’

  9. Genomic DNA Shearing/Sonication Subclone and Sequence Shotgun reads Assembly Contigs Finishing read Finishing Complete sequence

  10. Strategy Libraries Sequencing Assembly Closure Annotation Release Strategies for sequencing • Size and GC composition of genome • Volume of data • Ease of cloning • Ease of sequencing • Genome complexity • dispersed repetitive sequence • telomeres & centromeres • Politics/Funding

  11. Strategy Libraries Sequencing Assembly Closure Annotation Release Strategies: Clone by Clone • Simple (0.5 - 2 K reads) • Few problems with repeats • Relatively simple informatics • Scalability • Quality of physical map • Fingerprint / STS maps • End sequencing

  12. Strategy Libraries Sequencing Assembly Closure Annotation Release Strategies: Whole Chromosome shotgun (WCS) • Requires chromosome isolation • Moderate complexity (10’s K reads) • Problems with repeats • Complex informatics • Inefficient in isolation • Quality of physical map • Skims of mapped clones

  13. Strategy Libraries Sequencing Assembly Closure Annotation Release Strategies: Whole Genome shotgun (WGS) • Moderate to High complexity (10-100’s K reads) • Problems with repeats • Complex informatics • Quality of physical map • Fingerprint map • STS markers • End-sequences • Skims of mapped clones

  14. Strategy Libraries Sequencing Assembly Closure Annotation Release Sequencing my genome Politics Production Finishing Annotation TIME MONEY

  15. Strategy Libraries Sequencing Assembly Closure Annotation Release What do you get? DATA!!, DATA !!, and more DATA!! • Sequence • incomplete v complete • First-pass annotation • Gene discovery • Full annotation • A starting point for research

  16. ORFeome based functional genomics RNAi phenotypes Gene Knockout Expression Microarray Genome annotation is central to functional genomics

  17. Strategy Libraries Sequencing Assembly Closure Annotation Release Sequencing • Library construction • Colony picking • DNA preparation • Sequencing reactions • Electrophoresis • Tracking/Base calling

  18. Strategy Libraries Sequencing Assembly Closure Annotation Release Libraries • Essentially Sub-cloning • Generation of small insert libraries in a well characterised vector. • Ease of propagation • Ease of DNA purification • e.g. puc18, M13

  19. Strategy Libraries Sequencing Assembly Closure Annotation Release Libraries - testing • Simple concepts • Insert/Vector ratio • Real data • Insert size • Sequence …. • Simple analysis

  20. Strategy Libraries Sequencing Assembly Closure Annotation Release Sequence generation • Pick colonies • Template preparation • Sequence reactions • Standard terminator chemistry • pUC libraries sequenced with forward and reverse primers

  21. Strategy Libraries Sequencing Assembly Closure Annotation Release Sequence generation • Electrophoresis of products • Old style - slab gels, 32 > 64 > 96 lanes • New style - capillary gels, 96 lanes • Transfer of gel image to UNIX • Sequencing machines use a slave Mac/PC • Move data to centralised storage area for processing

  22. Strategy Libraries Sequencing Assembly Closure Annotation Release Gel image processing • Light-to-Dye estimation • Lane tracking • Lane editing • Trace extraction • Trace standardisation • Mobility correction • Background substitution

  23. Strategy Libraries Sequencing Assembly Closure Annotation Release Pre-processing • Base calling using Phred • modifies SCF file • Quality clipping • Vector clipping • Sequencing vector • Cloning vector • Screen for contaminants • Feature mark up (repeats/transposons)

  24. Strategy Libraries Sequencing Assembly Closure Annotation Release Finishing • Assembly: Process of taking raw single-pass reads into contiguous consensus sequence • Closure: Process of ordering and merging consensus sequences into a single contiguous sequence • Finished is defined as sequenced on both strands using multiple clones. In the absence of multiple clones the clone must be sequenced with multiple chemistries. The overall error rate is estimated at less than 1 error per 10 kb

  25. Strategy Libraries Sequencing Assembly Closure Annotation Release Genome Assembly • Pre-assembly • Assembly • Automated appraisal • Manual review

  26. Strategy Libraries Sequencing Assembly Closure Annotation Release Pre-Assembly • Convert to CAF format • flatfile text format • choice of assembler • choice of post-assembly modules • choice of assembly editor www.sanger.ac.uk/Software/CAF

  27. Strategy Libraries Sequencing Assembly Closure Annotation Release Assembly • Assemble using Phrap • Read fasta & quality scores from CAF file • Merge existing Phrap .ace file as necessary • Adjust clipping

  28. Strategy Libraries Sequencing Assembly Closure Annotation Release Assembly appraisal • auto-edit • removes 70% of read discrepancies • Remove cloning vector • Mark up sequence features • finish • Identify low-quality regions • Cover using ‘re-runs’ and ‘long-runs’ • Compare with current databases • plate contamination

  29. Strategy Libraries Sequencing Assembly Closure Annotation Release Manual Assembly appraisal • Use a sequence editor (GAP/consed) • Tools to identify Internal joins • Tools to identify and import data from an overlapping projects • Tools to check failed or mis-assembled reads for inclusion in project

  30. Strategy Libraries Sequencing Assembly Closure Annotation Release Manual editing • Sanger uses 100% edit strategy • Where additional data is required: • Check clipping • Additional sequencing • Template / Primer / Chemistry • Assemble new data into project • GAP4 Auto-assemble • Repeat whole process

  31. Strategy Libraries Sequencing Assembly Closure Annotation Release Manual Quality Checks • Force annotation tag consistency • All unedited data is re-assembled using Phrap • All high-quality discrepancies are reviewed • Confirm restriction digest (clones) • Check for inverted repeats • Manually check: • Areas of high-density edits • Areas with no supporting unedited data • Areas of low read coverage

  32. Strategy Libraries Sequencing Assembly Closure Annotation Release Gap closure • Read pairs • PCR reactions (long-range / combinatorial) • Small-insert libraries • Transposon-insertion libraries

  33. Strategy Libraries Sequencing Assembly Closure Annotation Release Gap closure - contig ordering • Read pair consistency • STS mapping • Physical mapping • Genetic mapping • Optical mapping • Large-insert clone • skims • end-sequencing

  34. Strategy Libraries Sequencing Assembly Closure Annotation Release Annotation • DNA features (repeats/similarities) • Gene finding • Peptide features • Initial role assignment • Others- regulatory regions

  35. Annotation of eukaryotic genomes Genomic DNA ab initio gene prediction transcription Unprocessed RNA RNA processing Mature mRNA Gm3 AAAAAAA Comparative gene prediction translation Nascent polypeptide folding Active enzyme Functional identification Function Reactant A Product B

  36. Genome analysis overview: C.elegans

  37. Strategy Libraries Sequencing Assembly Closure Annotation Release DNA features • Similarity features • mapping repeats • simple tandem and inverted • repeat families • mapping DNA similarities • EST/mRNAs in eukaryotes • Duplications, • RNAs • mapping peptide similarities • protein similarities

  38. Strategy Libraries Sequencing Assembly Closure Annotation Release Gene finding • ORF finding (simple but messy) • ab initio prediction • Measures of codon bias • Simple statistical frequencies • Comparative prediction • Using similarity data • Using cross-species similarities

  39. Strategy Libraries Sequencing Assembly Closure Annotation Release Peptide features • Peptide features • low-complexity regions • trans-membrane regions • structural information (coiled-coil) • Similarities and alignments • Protein families (InterPro/COGS)

  40. Strategy Libraries Sequencing Assembly Closure Annotation Release Initial role assignment • Simple attempt to describe the functional identity of a peptide • Uses data from: • peptide similarities • protein families • Vital for data mining • Large number of predicted genes remain hypothetical or unknown

  41. Strategy Libraries Sequencing Assembly Closure Annotation Release Other regulatory features • Ribosomal binding sites • Promoter regions

  42. Strategy Libraries Sequencing Assembly Closure Annotation Release Data Release • DNA release • Unfinished • Finished • Nucleotide databases • GENBANK/EMBL/DDBJ • Peptide databases • SWISSPROT/TREMBL/GENPEPT • Others

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