Monday, November 22, 2010

Retrieving transcriptome sequences for RNASeq analysis

One approach for analyzing RNASeq data from an organism with a well-annotated genome, is to align the reads to mRNA (cDNA) sequences instead of the genome. To do that you need to extract the transcript sequences from a database. This is how to extract ensembl transcript sequences from UCSC from within R:
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library(GenomicFeatures)
library(BSgenome.Hsapiens.UCSC.hg18)

tr <- makeTranscriptDbFromUCSC(genome="hg18", tablename="ensGene")
tr_seq <- extractTranscriptsFromGenome(Hsapiens, tr)
write.XStringSet(tr_seq, file="hg18.ensgene.transcripts.fasta", 'fasta', width=80, append=F)

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Next steps can be to build a reference index for bowtie, perform the alignment, and count the number of reads aligned in R using table(). Differential expression analysis may be performed by DESeq.




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