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

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

Nucleotide Database Growth

EMBL breakdown by organism

EMBL Release 65

Progress on Large Sequencing Projects

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

Genome size and sequencing strategies Genome size (log Mb) 1 2 3 4 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’

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

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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

Strategies: Whole Chromosome shotgun (WCS) Libraries Sequencing Release Assembly Annotation Closure Strategy 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

Strategies: Whole Genome shotgun (WGS) Libraries Sequencing Release Assembly Annotation Closure Strategy 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

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

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

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

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

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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

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

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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

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) Libraries Sequencing Release Assembly Annotation Closure Strategy

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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

Pre-Assembly Convert to CAF format flatfile text format Libraries Sequencing Release Assembly Annotation Closure Strategy 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

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

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

Manual Assembly appraisal Libraries Sequencing Release Assembly Annotation Closure Strategy 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

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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

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

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

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

Genome analysis overview: C.elegans

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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

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 Libraries Sequencing Release Assembly Annotation Closure Strategy

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

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