Genome sequencing and annotation Week 2 reading assignment - pages 63-78, 93-98, Boxes 2.1 and 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 – 32 P, 35 S –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

Cycle sequencing

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 kb fragments of DNA 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 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