SequencingProject - Sequencing project for Bi1x class...

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1 Sequencing project for Bi1x – class exercise Overview The main objective of this exercise is to show you a simplified version of the way microbiologists identify small subunit ribosomal RNA sequences (“16S” sequences) from the environment. You will learn how to (1) convert your sequencing traces to nucleotides (2) align the 16S sequences using the greengenes website (3) scan your sequences for chimeras, which you will eliminate from your database and (4) use MEGA to produce a phylogenetic tree of your sequences and their nearest neighbors (found by greengenes). You will then measure in MEGA the distance between your 16S sequences and the nearest neighbors and determine if they are of the same species, genus or phylum. Finally you will identify the precise taxonomic classification of the nearest neighbor for each of your 16S sequences and explain if given what’s known in the literature about the physiology of these isolates it’s plausible that you found these microbes in a fresh water pond (or could it be a contamination?). Please follow the instructions below and answer the questions in the bullets as you go along. Computer requirements: to complete this exercise you will need a (1) PC or a PC emulator on a Mac and (2) internet access. Part I: Learn a little more about the 16S gene The rrsA gene of E. coli codes for a small subunit ribosomal RNA (rRNA) gene. Let’s learn a little more about this gene. Go to the BioCyc microbial database (the default organism being E. coli ) In the search box type rrsA and click rrsA under genes . 1. What is the length of this gene? 2. Since you used ‘all bacterial primers’ for your PCR your primers should also match this rRNA gene. Find out the length of the amplicon you generated. The primer sequences you used were 357F (16S forward primer) 5’ CTCCTACGGGAGGCAGCAG 3’ 492RL2D (16S reverse primer) 5’ TACGGYTACCTTGTTACGACTT 3’ Here is a tool to help you reverse complement a nt sequence
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2 Hint 1. to get E. coli’s 16S sequence click on the rrsA hyperlink under Genes and then press the Nucleotide Sequence button . To convert this sequence into a long string of letters copy this sequence into notepad in order to remove non-text characters. Then copy this sequence into Word and replaces spaces “ “ with an empty string and then replace the newline character “^p” with an empty string. Save E. coli ’s sequence for later, we will be using it again for our tree. Hint 2. What is special about the nucleotide sequence of one of the primers above? Look at them carefully…no, it’s not a mistake…This website can perhaps help you: 3. What is the genomic context of this gene? Click on the Genome Browser button. You will see that rrsA is part of an operon. What other genes are present in this operon? What do they do? Is their function related to rrsA ? 4.
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SequencingProject - Sequencing project for Bi1x class...

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