Hi, Galaxy users!
I have a question on how to find out sense and antisense sequence.
I've got RNA seq data in the fastq format. The sequences inside are
partially complementary to each other (complementary is 10nt, while
entire is about 30nt). How can I separate these sequences into two
groups: sense and antisense (one thing I know is for the sense
sequence the 10th nucleotide is always "A")?
Thanks a lot!
Under the tool group "NGS: QC and manipulation" is a tool named
"Manipulate FASTQ". To filter for sequences containing an "A" at base
position 10 and remove them, use the settings are shown in the
.png. Also listed here:
Click on "Add new Match Reads"
Match Reads by: "Sequence Content"
Sequence Match Type: "Regular Expression", using
Click on "Add new Manipulate Reads"
Manipulate Reads on: "Miscellaneous Actions"
Miscellaneous Manipulation Type: "Remove Read"
This will result in the antisense reads being placed in the output,
minus any antisense that happened to have an A at the 10th base
position, which I am not sure is a concern or not.
To output the sense reads, or rather reads with an A at the 10 base
position, change the regular expression to be:
The logic is a bit backwards - you are filtering for what you will be
removing - the opposite will be in the output.
Hopefully this helps! Peter's advice about aligning to the genome and
determining strand/orientation vs known transcripts from the results
mostly likely your second best choice (and more complicated). Tools in
"Interval Operation" group will be of help if you go down that path.
Depending on how your sequences were prepared, you might be able to
look for a poly-A tail as a clue to orientation. Another approach is
compare the (assembled) transcripts to known genes and if you only
get matches on one strand that is probably the correct orientation.
Why is that? Is this related to your library preparation?