How can we find genes?

Search for themLook them up

How do I get from this…

>mouse_ear_cress_1080 GAAATAATCAATGGAATATGTAGAGGTCTCCTGTACCTTCACAGAGATTCTAGGCTGAGAGCAGTGCATATAGATATCTTTCGTACTCATCTGCTTTTTCTGGTCTCCATCACAAAAGCCAACTAGGTAATCATATCAATCTCTCTTTACCGTTTACTCGACCTTTTCCAATCAGGTGCT TCTGGTGTGTCTACTACTATCAGTTTTAGGTCTTTGTATACCTGATCTTATCTGCTACTG AGGCTTGTAAAAGTGATTAAAACTGTGACATTTACTCTAAGAGAAGTAACCTGTTTGATGCATTTCCCTAATATACCGGTGTGGAAAAGTGTAGGTATCTGTACTCAGCTGAAATGGTGGACGATTTTGAAGAAGATGAACTCTCATTGACTGAAAGCGGGTTGAAGAGTGAAGATGGCGTTATTATCGAGATGAATGTCTCCTGGATGCTTTTATTATCATGTTTGGGAATTTACCAAGGGAGAGGTATCAGAATCTATCTTAGAAGGTTACATTTAGCTCAAGCTTGCATCAACATCTTTACTTAGAGCTCTACGGGTTTTAGTGTGTTTGAAGTTTCTTAACTCCTAGTATAATTAGAATCTTCTGCAGCAGACTTTAGAGTTTTGGGATGTAGAGCTAACCAGAGTCGGTTTGTTTAAACTAGAATCTTTTTATGTAGCAGACTTGTTCAGTACCTGAATACCAGTTTTAAATTACCGTCAGATGTTGATCTTGTTGGTAATAATGGAGAAACGGAAGAATAATTAGACGAAACAAACTCTTTAAGAACGTATCTTTCAGTTTTCCATCACAAATTTTCTTACAAGCTACAAAAATCGAACTATATATAACTGAACCGAATTTAAACCGGAGGGAGGGTTTGACTTTGGTCAATCACATTTCCAATGATACCGTCGTTTGGTTTGGGGAAGCCTCGTCGTACAAATACGACGTCGTTTAAGGAAAGCCCTCCTTAACCCCAGTTATAAGCTCAAAGTTGTACTTGACCTTTTTAAAGAAGCACGAAACGAAAAACCCTAAAATTCCCAAGCAGAGAAAGAGAGACAGAGCAAGTACAGATTTCAACTAGCTCAAGATGATCATCCCTGTTCGTTGCTTTACTTGTGGAAAGGTTGATATTTTCCCCTTCGCTTTGGTCTTATTTAGGGTTTTACTCCGTCTTTATAGGGTTTTAGTTACTCCAAATTTGGCTAAGAAGAGATCTTTACTCTCTGTATTTGACACGAATGTTTTTAATCGGTTGGATACATGTTGGGTCGATTAGAGAAATAAAGTATTGAGCTTTACTAAGCTTTCACCTTGTGATTGGTTTAGGTGATTGGAAACAAATGGGATCAGTATCTTGATCTTCTCCAGCTCGACTACACTGAAGGGTAAGCTTACAATGATTCTCACTTCTTGCTGCTCTAATCATCATACTTTGTGTCAAAAAGAGAGTAATTGCTTTGCGTTTTAGAGAAATTAGCCCAGATTTCGTATTGGGTCTGTGAAGTTTCATATTAGCTAACACACTTCTCTAATTGATAACAGAAGCTATAAAATAGATTTGCTGATGAAGGAGTTAGCTTTTTATAATCTTCTGTGTTTGTGTTTTACTGTCTGTGTCATTGGAAGAGACTATGTCCTGCCTATATAATCTCTATGTGCCTATCTAGATTTTCTATACAATTGATATTTGATAGAAGTAGAAAGTAAGACTTAAGGTCTTTTGATTAGACTTGTGCCCATCTACATGATTCTTATTGGACTAATCATTCTTTGTGTGAAAATAGAATACTTTGTCTGAACATGAGAGAATGGTTCATAATACGTGTGAAGTATGGGATTAGTTCAACAATTTCGCTATTGGAGAAGCAAACCAAGGGTTAATCGTTTATAGGGTTAAGCTAATGCTCTGCTCTTTATATGTTATTGGAACAGACTATTGTTGTGCCTATCTTGTTTAGTTGTAGATTCTATCTCGACTGTTATAAGTATGACTGAAGGCTTGATGACTTATGATTCTCTTTACACCTGTAGAAGGATTTAAGCTTGGTGTCTAGATATTCAATCTGTGTTGGTTTTGTCTTTCTTTTGGCTCTTAGTGTTGTTCAATCTCCTCAATAGGTATGAAGTTACAATATCCTTATTATTTTGCAGGGACGCACTTGATGCACTCCAGCTAGTCAGATACTGCTGCAGGCGTATGCTAATGACCTTGCATCAACATCTTTACTTAGAGCTCTACGGGTTTTAGTGTGT

…to this?

Meaning?

Mathematical Tools (Code; statistics)

Comparative Tools (Database searches)

What do we know about genes?• Expressed (Transcribed)

– Transcriptional start & termination sites (TXSS, TXTS)– Transcription artefacts (cDNA & ESTs)

• Regulated– Promoters (TATAAA)– Transcription Factor Binding Sites– CpG (Cytosin methylation)

• Meaningful (Translated)– 3n basepairs– Codon usage– Translational start & stop/termination codons (TLSS, TLTS)– Translation artefacts (proteins)

• Spliced– Splice sites (GT-AG)

• Derived (Homology: Paralogy/Orthology)– Search for known genes, proteins (BLAST)

How might this knowledge help to find genes?

• Predict genes– Look for potential starts and stops.– Connect them into open reading frames (ORFs).– Filter for “correct’ length & codon usage.

• Search databases– Known genes: UniGene– Known proteins: UniProt

• Use transcript evidence– cDNA– ESTs– proteins

Operating computationally

• Go to beginning of sequence start SCAN• If ATG register putative TLSS; then

– Move in 3-steps & count steps (=COUNTS)– If 3-step = (TAA or TAG or TGA), register putative TLTS– If register evaluate COUNTS (= triplets)

If COUNTS < minimum discard; then go behind ATG above and start SCAN

If COUNTS > maximum discard; then go behind ATG above and start SCAN

If minimum < COUNTS < maximum record as GENE with TLSS, TLTS; then go behind ATG above and start SCAN.

• Arrive at end of sequence stop SCAN

Find gene families

Mathematical evidence

Analyze large data

sets

Browse in ccontext

Construct gene

models

Annotation workflow

Biological evidence

Browse results

Get/Generate sequence

Annotation Cheat Sheet• Open existing project or generate new (Red square)

• Run RepeatMasker

• Generate evidence (Predictions, BLAST searches)

• Synthesize evidence into gene models (Apollo)

• Browse results locally and in context (Phytozome)

• Conduct functional analysis (link from Browser)

• Prospect for gene family (Yellow Line from Browser)• Select region that holds biological gene evidence

• Optimize work space and zoom to region (View tab)

• Expand all tiers (Tiers tab)

• Drag evidence item(s) onto workspace (mouse)

• Edit to match biol. evidence (right-click item for tools)

• Record what was done in Annotation Info Editor

• Assess necessity to build alternative model(s)

• Upload model(s) to DNA Subway (File tab)

A. DNA Subway

B. Apollo

Predictors (mathematical evidence)

• Utilize predominantly mathematical methods (statistical).• Search for patterns

– Some score starts, stops, splice sites (GenScan).– Some score nucleotides (Augustus, FGenesH).

• Few incorporate EST data and/or known genes/proteins.• Require optimization for each new species (training).• Accuracy:

– False positives (scoring non-genes as genes):5% - 50%.– False negatives (missed genes): 5%-40%.– Weak or unable in determining first and last exons, and UTRs.

• Specific for gene models (spliced genes, non-spliced genes).• Specialty predictors (tRNA Scan, RepeatMasker).

Search tools (biological evidence)

• Search sequence (molecules; tangible) databases:– Known genes– Known proteins– cDNAs & ESTs

• Utilize alignment methods (BLAST, BLAT).• Reliability:

– Good in determining gene locations and general gene structures.– Weak in exactly determining exon/intron borders.– Unlikely to correctly determine TXSS and TXTS.– Should be used with cDNA/EST from same species as genome.

Sequence & course material repository

http://gfx.dnalc.org/files/evidenceDon’t open items, save them to your computer!!

• Annotation (sequences & evidence)• Manuals (DNA, Subway, Apollo, JalView)• Presentations (.ppt files)• Prospecting (sequences)• Readings (Bioinformatics tools, splicing, etc.)• Worksheets (Word docs, handouts, etc.)• BCR-ABL (temporary; not course-related)