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Common OTUs across sample using mothur

November 23, 2011 Leave a comment

What do i have?
Three technical replicates of 16S amplification from a sample.
What do I intend to do?
Test the goodness of the reproducibility.
How am i going to do it?
Based on the Zhou et al. 2011 ISMEJ paper, I will compare the common OTUs across all the samples. At the end, I want to make a venn diagram that represents the number of OTUs that are common across the sample?
Let it begin……
I will use mothur v 1.22 to do this. Lets name my sample as A1, A2, and A3. I got a fasta file for each sample named: A1.fasta, A2.fasta, and A3.fasta. The sequences were already filtered to remove erroneous and chimeric reads. Additionally, they were trimmed to same region, and then aligned and filtered(remove the column-gaps from alignment) as well.
First, I created a group file. Its a two column file, which contains the name of the sequence in the first column and the sample name in the second column. For example:
G45667889 A1
G47879890 A2
G45454800 A3
G5803i808 A1
There is a command in mothur called make.group, which creates this file, if you had not created it during demultiplexing step.

make.group(fasta=A1.fasta-A2.fasta-A3.fasta,group=A1-A2-A3)

It will create a file called merge.groups, which will be used extensively for downstream analysis.

Since, we are going to find the common OTUs across samples, we will combine all the fasta using cat.

cat A1.fasta A2.fasta A3.fasta > A123.fasta

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