Chapter 9: Sequence Comparisons

Most sequencing methods rely on shotgun sequencing: do a partial digest of fragment, get reads and using computational methods overlap the reads and get the entire sequence

Clone by Clone Approach

Using genetic mapping techniques, sequences large genome by ordering sub clones

  • Many copies of the genome are fragmented in partial digest and the large fragments are cloned into BACs and amplified, clones are purified for further analysis and completely digested, chosen to produce a patter of small fragments for each clone
  • Comparison of the patterns reveal overlap regions allowing them to be lined up and individual BAC clones are then sheared into smaller fragments and cloned the resulting sub clones are sequenced to ensure each part of original clone is analyzed several times
  • Sequence of individual BAC clones are assembled using physical map as guide and entire genome is assembles
  • Used when we did not have computational tools, time consuming but precise
  • Difficult to do with repetitive DNA, some regions cannot be cloned (code lethal protein)

Whole Genome Shotgun Sequencing

Cheaper initially, easier (lot of bioinformatics analysis) worse for repetition

  • Prepare small clones directly from gDNA, set of overlapping clones 2-10kb and fragments were then selected and sequenced randomly, sequence was assembled with the aid of super computer (overlapping regions)
  • Reads (small size) sequenced into contigs (set of overlapping DNA) sequenced into scaffolds

Sequence Analysis

Blast: Basic Local Alignment Search Tool, BLAST query into database and finds similar sections

  • E Value: Number of sequences expected by chance when searching database of size

MSA: Multiple sequence alignments, to compare conserved and variable sequences

Phylogenetic Trees: Identify organisms with high degree of evolutionary similarity

GWAS: Genome wide association studies, compare wild type to mutant to find certain gene: confirm

Ab Initio: Predict novel protein products based on features from the sequence (easier for prokaryotes because ORF is in 1 section without introns and highly conserved -10 to -35 contain putative genes)

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