1 / 11

Genome sequencing and annotation

Genome sequencing and annotation. Week 2 reading assignment - pages 63-78, 93-98, 105-111. Boxes 2.1 and 2.3 - don’t worry about details of similarity scoring and hidden markov models. Sequencing - dideoxy method for DNA sequencing. Methods for sequencing genomes.

karena
Download Presentation

Genome sequencing and annotation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Genome sequencing and annotation Week 2 reading assignment - pages 63-78, 93-98, 105-111. Boxes 2.1 and 2.3 - don’t worry about details of similarity scoring and hidden markov models.

  2. Sequencing - dideoxy method for DNA sequencing. • Methods for sequencing genomes. • Methods for finding and annotating genes in microbial genomes.

  3. Dideoxy sequencing (Sanger method) • Developed by Frederick Sanger (for which he won his second Nobel Prize in 1980).

  4. Two types of labeling • Radioactive • 32P, 35S • Run out each dideoxy base in a separate reaction, lane on a gel. • No longer used • Fluorescent • Four different fluorophores for each base • Can be mixed. • Chromatograms - GTSF

  5. Cycle sequencing

  6. Phred • Method for automated quality assessment of DNA sequence traces. • Variance in peak spacing in 7 peak window • Ratio of largest uncalled peak to smallest called peak in 7 and 3 peak windows. • Number of bases between current base and nearest unresolved base. • Phred score = 10 x (-log(P)). • Phred scores of 20 or higher are considered good calls. Why?

  7. Sequencing of genomes • Hierarchical or contig based sequencing • Clone smaller seqments of the genome. • Labor intensive, slow • Not needed for sequencing microbial genomes • Shotgun method • Randomly clone and sequence 1.5-2 kb fragments of DNA. 5-10 fold coverage. • Computationally intensive.

  8. Sequence assembly • Focus of this week’s lab exercise • Algorithms to align and edit multiple sequences • Phrap and Consed • Sequencher (commercial) for lab. • List of features of good sequence editors on page 70.

  9. Sequenced microbial genomes • Haemophilus influenzae - 1995. • Over 250 microbes currently sequenced. • Why sequence so many microbial genomes? • Develop technology for human genome project • Examine the genomes of a wide range of microbes • Novel drug/vaccine targets • Identify new agricultural and industrial important enzymes • Comparative genomics/evolution • Sequence microbes that have no genetics • Sequence microbes we can’t culture - metagenomics • Because we can. • Financially feasible to sequence entire genomes.

  10. Finding functional features in a microbial genome. • Genes • Origin of replication • rRNA operons, tRNAs • Promoters • Transcription terminators • Horizontially transferred DNA • GC content

More Related