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Alignment of each of these 100 sets of simulated short reads to the database of repeats gives the expected outcome if each repeat class in the genome were transcribed at random.
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The BWA program (version 0.6.1-r104) was used to match-cleaned reads to the Human Ensembl database and the exon junction database for annotation successively, with the allowance of 5 mismatches for each read (Li and Durbin 2009).
Alternatively, the remaining reads can be aligned on a laptop using BLASTn to just the viral fraction of the NT database, followed by BLASTn alignment of the viral reads to the NT database to verify that they are correctly identified.
Prior to mapping reads to the reference database, raw reads were transformed into clean reads as earlier described.
We mapped RNA-seq reads to the reduced database using BWA [ 27] with default settings.
The alignment of the reads to the reference database is probably the most important step of the analysis workflow.
We attempted to use the LAST software to align nanopore reads to the NT database (June 2014, ~60 Gb in size).
Prior to mapping reads to the reference database, we filtered all sequences to remove low quality sequences.
We mapped the filtered Illumina reads to the reduced database using BWA [ 57] as implemented in the Galaxy Project [ 58].
In both cases, we were able to efficiently map large amounts of the reads to the reference database and to calculate evidence values per function.
Furthermore, we sought to determine whether our RNA-seq assay could detect novel splice isoforms simply by mapping the unique reads to the junction database built in house.
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