I have obtained (non-annotated) long non-coding RNA candidates from transcriptome data. I have the chromosomal positions of the candidates. I would like to retrieve the sequences for these candidates, in order to run experiments (eg, RNAi). Any advice on how to do this would be appreciated.
Thanks.
2 answers
Hi Eric,
you can use UCSC genome browser.
For batch processing you can read the FAQ`s or use Galaxy.
I hope this helps.
If you're into programming (and R (and bioconductor)), let's assume you're in human:
## Setting the environment:
library(BSgenome.Hsapiens.UCSC.hg19)
library(GenomicRanges)
## Let's say these are some of the genomic coordinates
## of your non-coding RNAs:
coords <- GRanges(c('chr1', 'chr2', 'chr3'),
IRanges(c(1e7, 2e7, 3e7), width=100),
c('+', '-', '+'))
## which looks like:
coords
GRanges with 3 ranges and 0 elementMetadata values:
seqnames ranges strand
<Rle> <IRanges> <Rle>
[1] chr1 [10000000, 10000099] +
[2] chr2 [20000000, 20000099] -
[3] chr3 [30000000, 30000099] +
## There is an `Hsapiens` object loaded into your workspace
## which got there by loading the
## BSgenome.Hsapiens.UCSC.hg19 library, you can combine a
## GRanges object with `Hsapiens` to easily fetch the seqences
seqs <- getSeq(Hsapiens, coords)
## Which look like:
seqs
A DNAStringSet instance of length 3
width seq
[1] 100 AACCCCGTCTCTACAATAAATTAAAATATTAGCT...CTTGGCGGGCTGAGGTGGGAGAATCATCCAAGC
[2] 100 GCAGGTTTGGAATCTGTTTCATGAAAACTCCCAG...TCATCCATTTGCATAGCATCAAATGGAAATGTC
[3] 100 ATGTTTGATTGATGCCCTCGAACTTGGCTAGTTG...AAACTCAATTGAATTATATTGCCTTAGCTTATA
While convenient, the getSeq function might require more ram than iterating over the coords of your loci one chromosome at a time, but I'll leave those implementation details as an assignment for the reader ;-)
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