Hi all I want to get corresponding mRNA sequences against 22283 probset ids of HG-U133A platform of affymetrix microarray experiment. But I have not been able to get that. I have mapped all those ids to biomart but not got all the sequences of transcripts.
Looking forward to reply.
Thanks
Monu Kumar
For what is worth, here is a step by step guide of how to do this with biomart :
1) connect to biomart
2) In list "CHOOSE DATABASE" -> select "Ensembl Genes 66"
3) In list "CHOOSE DATASET" -> select "Homo sapiens genes (GRCh37.p6)"
4) Then on the left, clik on "Filter"
5) In the main panel clink on Genes
6) Tick the checkbox "Limit to genes ... " and select in the listbox "with affymetrix microarray hg u133a 2 probeset ID(s)"
7) Then on the left, click on "Attributes"
8) In the main panel, check the radio button in front of "Sequences"
9) Just below, click on the "+" box in front of "sequences", and then select the type of sequence yo want, for example "cDNA sequences"
10) Facultative : Just below, click on the "+" box in front of "Header information". By default, you will have Ensembl Gene ID and Ensembl Transcript ID cheked, you may want to change this
11) Then in the upper panel, click on "RESULTS", you will see the begining of what you want.
12) To export all sequences in a text file, choose your file format (default is FASTA), You SHOULD tick the box in front of UNIQUE RESULTS ONLY, and then click on "GO"
What I just described can be accessed directely with this link
Thanks a lot for your valuable help. I have gone through the steps and got 4330 sequences in total. But I am having bit confusion. For 22283 probset ids, should not there be 22283 mRNA (cDNA) sequences in returns (if I donot check the box corresponding to unique results only)? I am getting same 4330 sequences for unique as well as otherwise. What my perception is that for 22283 IDs, I should get 22283 mRNA/cDNA sequences (right now I am not concerened about redundant ones). I need whole sequences. So can you suggest something ?
Thanking you .
Monu Kumar
The short answer is: no, there is no reason why 22 283 probeset IDs should have a one-to-one relation with a transcript sequence. Probesets may, variously, map to a unique transcript, several transcripts, many probesets to one transcript or no transcript at all.
It's best not to make assumptions and inspect the annotation files for the microarray platform that you are using. If you're using R/Bioconductor, you can install the HG-U133A annotation package:
source("http://bioconductor.org/biocLite.R")
biocLite("hgu133a.db")
And then, for example:
entrez <- toTable(hgu133aENTREZID)
dim(entrez)
# [1] 20373 2
length(unique(entrez$gene_id))
# [1] 12704
Showing that only 20 373 of the probesets map to Entrez Gene IDs and uniquely, to only 12 704.
Or: you can visit the Affymetrix product page for HG-U133, click on Technical Documentation and download one of the many annotation files from there. It's not always obvious which is the most useful, so you may have to explore a little.
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When you say "mapped all those ids to biomart" what exactly did you do? Use HG-U133A probeset IDs as filters and try to retrieve transcripts IDs/sequences as attributes?
Hello Yes I have used these probset IDs as filters, but I am not getting exact number of sequences of mRNAs as those of input IDs. I was expecting 22283 mRNA sequences in return, but that was not the case. Right now I am not bothering about redundant mRNAs. What I expect is one to one returns for given query. Is it possible or am I expecting something wrong ?
You're expecting something wrong. Let me work on an answer...