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. • Methods for finding and annotating genes in microbial genomes.
Dideoxy sequencing (Sanger method) • Developed by Frederick Sanger (for which he won his second Nobel Prize in 1980).
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
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?
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.
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.
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.
Finding functional features in a microbial genome. • Genes • Origin of replication • rRNA operons, tRNAs • Promoters • Transcription terminators • Horizontially transferred DNA • GC content