1. UCSC Genome Browser Zeevik Melamed & Dror Hollander Gil Ast Lab Sackler Medical School

2. Understanding the Genome DNA RNA protein miRNA gene expression non-coding RNA secondary structure alternative splicing exon-intron structure histone midifications genes GC content promoters repetitive elements conservation SNPs nucleosome occupancy How can you examine a genomic segment while taking all of these factors into account?

3. Lecture Overview  UCSC Genome Browser Interface & selected tracks Detecting alternative splicing events BLAT PCR  Galaxy

4. UCSC Genome Browser  Basic design: “the Genome Browser stacks annotation tracks beneath genome coordinate positions, allowing rapid visual correlation of different types of information” genome track (64 eukaryote genomes)

5. Genome Browsing…

6. Basic Genome Browser Interface UTR CDS intron gene direction (> / <) chromosomal position genomic coordinates zoom Refseq Genes track UCSC Genes track mark and drag here to zoom in Black - feature has a corresponding entry in the Protein Data Bank (PDB) Dark blue - transcript has been reviewed or validated by either the RefSeq, SwissProt or CCDS staff Medium blue - other RefSeq transcripts Light blue - non-RefSeq transcripts start codons in green stop codons in red

7. Basic Genome Browser Interface  Configure track visualization:

8. Basic Genome Browser Interface  “RefSeq track shows known human protein-coding and non-protein-coding genes taken from the NCBI RNA reference sequences collection (RefSeq). The data are updated daily”  “The UCSC track shows gene predictions based on data from RefSeq, Genbank, CCDS and UniProt… includes both protein-coding and putative non-coding transcripts… Compared to RefSeq, this gene set has generally about 10% more protein-coding genes, approximately five times as many putative non-coding genes, and about twice as many splice variants” Let’s examine a few examples online…

9. Basic Genome Browser Interface  A few more tracks worth mentioning: miRNA (Genes and Gene Prediction Tracks -> sno/miRNA) conservation (Comparative Genomics -> Conservation) Expression tracks Regulation tracks (chromatin structure and modifications, DNA methylation, etc.; includes ENCODE data) RNA secondary structure (Genes and Gene Prediction Tracks -> EvoFold) SNPs (Variation and Repeats -> SNPs)

10. Basic Genome Browser Interface  Convert browser window to an image file:  Get genomic DNA for the viewed coordinates:  Convert sequence to a different genome assembly or genome:

11. Lecture Overview  UCSC Genome Browser Interface & selected tracks Detecting alternative splicing events BLAT PCR  Galaxy

12. Detecting Alternative Splicing Events  Via Human mRNAs & Spliced ESTs tracks (mRNA and EST Tracks) gene DNA mRNA “The mRNA track shows alignments between human mRNAs in GenBank and the genome” “…alignments between human expressed sequence tags (ESTs) in GenBank and the genome… ESTs are single-read sequences, typically about 500 bases in length”

13. Detecting Alternative Splicing Events  Via Alt Events track (Genes and Gene Prediction Tracks) – based on UCSC genes gene DNA > cassetteExon

14. Detecting Alternative Splicing Events  Via Burge RNA-seq track (Expression) Click on the track name to choose tissues gene DNA Burge RNA-seq

15. Different Alternative Splicing Types  Exon skipping Exon skipping  Alternative splice site (3’) Alternative splice site (3’)  Intron retention Intron retention

16. Lecture Overview  UCSC Genome Browser Interface & selected tracks Detecting alternative splicing events BLAT PCR  Galaxy

17. BLAT  BLAT = Blast-Like Alignment Tool  BLAT is designed to find similarity of >95% on DNA and >80% for protein BLAT query

18. Lecture Overview  UCSC Genome Browser Interface & selected tracks Detecting alternative splicing events BLAT PCR  Galaxy

19. PCR temperatures amplicon in fasta format coordinatesstrandprimers amplicon

20. Lecture Overview  UCSC Genome Browser Interface & selected tracks Detecting alternative splicing events BLAT PCR  Galaxy

21. Galaxy  “Galaxy allows you to do analyses you cannot do anywhere else without the need to install or download anything. You can analyze multiple alignemnts, compare genomic annotations, profile metagenomic samples and much much more...”

22. Galaxy – What Is It Good for?  Getting the best out of UCSC Operating on UCSC data  Supports operations both at the interval level, and at the sequence level  Designed for biologists!

23. Galaxy – Typical Workflow  Extract sets of coordinates either upload from computer or from UCSC table browser  Operate on different sets of coordinates (intersect, subtract etc.)  Fetch genomic sequences of coordinates