As I was thinking about subsampling reads to make more fair comparisons I realized that there is probably still at least a small proportion of contaminating rRNA reads left in the transcriptomes. If I subsample before filtering out those reads, I may be losing out of data mapping to genes I care about downstream. In order to avoid this I decided to map the reads with Bowtie to the SILVA rRNA gene database and use only those reads that fail to find a match for anything later on in the pipeline.
I started by getting the non-redundant full length sequences from release 123 off of the mothur site. This file has sequences from bacteria (152308), eukaryotes (16209), and archea (3901). It looks like this:
1 2 3 4 AB006051.LobTirol ......................................................ATG---------CGTACGCCATGCGTG------- GGCTAGACT------CGTAC------------------------------ACGTACGACACGTCCATAGACTCGATAC---------- ...
In order to align reads to this file, I needed to remove the gaps before making the Bowtie database. I used the mothur command that removes gaps as follows:
This gives you a fasta file with just sequence names and nucleic acids remaining, no more gaps. Making the bowtie database is almost as simple with the follow command:
1 /home/mljenior/bin/bowtie/bowtie-build silva.nr_v123.ng.fasta silva_db
Now it’s ready to be used for filtering out rRNA reads from my metatranscriptomes. I’ll post the results from how well it does probably later this week.