20.109(S14):Phylogenetic and primer analyses (Day7)

Introduction

Molecular phylogeny, or phylogenetics, is used to study relationships among organisms. The most common approach these days involves examining nucleic acid sequences or protein data from specific genetic loci; frequently the goal is to define data down to the species level. All life forms on earth trace back to a few organisms that lived billions of years ago and all share a common descent. Groups of organisms that are closely related to each other diverged from more recent shared common ancestors. Phylogeny remains one of the only effective means of describing these relationships, which can be difficult to assess by other means.

The goals of phylogenetics are to 1) reconstruct the correct genealogical relationship between organisms/genes/sequence data and 2) to estimate their divergence since sharing a common ancestor. The process of phylogenetic reconstruction relies heavily on correct comparison of the traits under question, whether it is morphological data (such as wing lengths) or sequence data. For sequence data, comparison is made by the alignment of a set of orthologous sequences, which we will do in lab from the 16s rRNA gene.

Today, we have a choice of algorithms (distance-based, neighbor-joining, parsimony, likelihood, and other) for reconstructing a phylogenetic tree that depicts the relationships among aligned sequences. A number of models for defining how the mutations between sequences (genetic substitution) are assessed are also available. Each of these methods and models has advantages and disadvantages, which are closely considered (ideally!) in any formal published phylogenetics study. In the world of microbial community analysis, a popular choice is the neighbor-joining method (Saitou and Nei, 1987), which is one of the methods that deals most accurately and consistently with large data sets. Regardless of the best method, however, the result -- a reconstructed phylogenetic tree -- has proven to be an extremely useful qualitative and often even quantitative tool for examining the relationships among organisms.

EXPAND

Somewheres here? Reconnect to goal. Or earlier?: We might ask: If two microbiomes are phylogenetically different, but functionally equivalent, does that mean they will be susceptible or resistant to similar pathogens? What do differences in microbiome structure mean for a bird’s ability to carry influenza virus, microsporidia, giardia species, or other gull associated microbes?

Protocols

For next time

Some of you have journal clubs next time. No other required homework is due on Day 8.

1. The following bonus assignment may be submitted on Day 8: Prepare a figure and caption for your primer design summary that shows your raw PCR results -- the agarose gel. (Later you might decide to process this data in some way, but not necessarily.) Write an early draft of the accompanying main text paragraph.

Reagent list

• Mostly your brains!

• Agarose gels
  • 2:1 mixture of high-resolution:standard agarose
  • Prepared in TAE buffer
  • With SYBR Safe stain (Invitrogen)
    • used at manufacturer's recommended concentration, 10000-fold dilution
• NEB loading dye (6X stock)
• Gels made and run in 1X TAE buffer
  • 40 mM Tris
  • 20 mM Acetic Acid
  • 1 mM EDTA, pH 8.3
• 100 bp DNA ladder from New England BioLabs