Analysis of sequence data is no small task. “Sequence gazing” can swallow hours of time with little or no results. There are also many web-based programs to decipher patterns. The nucleotide or its translated protein can be examined in this way. Thanks to the genome sequence information that is now available, a new verb, “to BLAST,” has been coined to describe the comparison of your own sequence to sequences from other organisms. BLAST is an acronym for Basic Local Alignment Search Tool, and can be accessed through the National Center for Biotechnology Information (NCBI) home page. You will use BLAST to identify which bacterial populations are present in your bird cloacal samples. These identifications will be used to complete phylogenetics and assess population differences that may be present in the microbiomes of birds based on sex or location.
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.
Bird microbiome analysis
You will take several steps to analyze your bird stool sequencing data with your partner and ultimately across the entire class. Below are links to the relevant steps that you practiced last time. Remember, you'll need to finish analyzing your sequences today so that your collaborators can have access to all of the data!
- For each of your clones, you will trim and combine the forward and reverse sequencing results to get one intact 16S rRNA gene sequence.
- For each sequence, you will use BLAST to determine the closest known bacterial species to that sequence.
- You will post the sequences and a summary of the species that you found, according to a specific template. See Part D from M1D7.
- You should then team up with your partner and together, you will align all of the robust sequences from your bird sample, up to 24 of them, in a program called MEGA, and subsequently construct a phylogenetic tree following Part E from M1D7.
- Each group must post a complete alignment file and tree for their bird sample.
- These trees will be used to make cross-class comparisons, along with composite trees for each bird population. (See assignment description for further guidance.)
- Make sure use all the tools available to you -- for example, complete an UniFrac analysis to quantitatively compare your bird samples and determine if geography or sex play a role in shaping the microbiome.
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