Find out sense and antisense sequences
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! Best, Zhiqiang
On Mon, Nov 26, 2012 at 6:47 PM, Zhiqiang Shu <zshu@bio.fsu.edu> wrote:
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
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 to compare the (assembled) transcripts to known genes and if you only get matches on one strand that is probably the correct orientation.
(one thing I know is for the sense sequence the 10th nucleotide is always "A")?
Why is that? Is this related to your library preparation? Peter
Thank you, Peter! I think I should put more detailed information here. What I'm doing is piRNA data. Two groups of piRNA (named sense and antisense)are in the library. As I said, they are complementary to each other for about 10 nt, while the whole length is about 30nt. For the sense group, they share the feature of having an "A" at their 10th. In this case, how can I deal with it? One possible way come up is inverting all sequences and aligning them. Thanks! Best, Zhiqiang Quoting "Peter Cock" <p.j.a.cock@googlemail.com>:
On Mon, Nov 26, 2012 at 6:47 PM, Zhiqiang Shu <zshu@bio.fsu.edu> wrote:
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
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 to compare the (assembled) transcripts to known genes and if you only get matches on one strand that is probably the correct orientation.
(one thing I know is for the sense sequence the 10th nucleotide is always "A")?
Why is that? Is this related to your library preparation?
Peter
-------------------------- Zhiqiang Shu/Deng Lab Department of Biological Science Florida State University 319 Stadium Dr. Tallahassee, FL, USA, 32306-4295 zshu@bio.fsu.edu
When do you need them and are you likely to need more in the future? We used the TruSeq whole exome kits, though we plan to switch to Agilent. Currently using Agilent's Kinome kit, would have them available. Agilent give substantial volume discount Richard B. Everson, MD, MPH Deputy Director for Cancer Prevention and Control Carole and Ray Neag Comprehensive Cancer Center University of Connecticut Health Center 263 Farmington Ave MC 1628 Farmington, Connecticut 06030-2875 email: everson@uchc.edu Phone: (860) 679-6055 Fax: Pt (860) 679-4815 Admin (860) 679-4451 -----Original Message----- From: galaxy-user-bounces@lists.bx.psu.edu [mailto:galaxy-user-bounces@lists.bx.psu.edu] On Behalf Of Zhiqiang Shu Sent: Monday, November 26, 2012 3:29 PM To: Peter Cock Cc: Galaxy-user Subject: Re: [galaxy-user] Find out sense and antisense sequences Thank you, Peter! I think I should put more detailed information here. What I'm doing is piRNA data. Two groups of piRNA (named sense and antisense)are in the library. As I said, they are complementary to each other for about 10 nt, while the whole length is about 30nt. For the sense group, they share the feature of having an "A" at their 10th. In this case, how can I deal with it? One possible way come up is inverting all sequences and aligning them. Thanks! Best, Zhiqiang Quoting "Peter Cock" <p.j.a.cock@googlemail.com>:
On Mon, Nov 26, 2012 at 6:47 PM, Zhiqiang Shu <zshu@bio.fsu.edu> wrote:
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
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 to compare the (assembled) transcripts to known genes and if you only get matches on one strand that is probably the correct orientation.
(one thing I know is for the sense sequence the 10th nucleotide is always "A")?
Why is that? Is this related to your library preparation?
Peter
-------------------------- Zhiqiang Shu/Deng Lab Department of Biological Science Florida State University 319 Stadium Dr. Tallahassee, FL, USA, 32306-4295 zshu@bio.fsu.edu ___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/
Hi Zhiqiang, 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 attatched .png. Also listed here: Click on "Add new Match Reads" Match Reads by: "Sequence Content" Sequence Match Type: "Regular Expression", using ^.{9}A.+ 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: ^.{9}[^A].+ 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 is mostly likely your second best choice (and more complicated). Tools in "Interval Operation" group will be of help if you go down that path. Best, Jen Galaxy team On 11/26/12 10:47 AM, Zhiqiang Shu wrote:
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!
Best, Zhiqiang
___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:
http://lists.bx.psu.edu/listinfo/galaxy-dev
To manage your subscriptions to this and other Galaxy lists, please use the interface at:
-- Jennifer Jackson http://galaxyproject.org
Thank you so much, Jennifer! Your method is pretty good for filtering the sequences that have "A" at the 10th base position.But I'm sure if it have employed 10 bp complementary criterion? For the antisense, the 5' will be T (in complementary to A). Besides, my library contains other small RNAs. That's why I concern output files of sense and antisense must have 10bp complementary. Another quick question, in you regular expression, what does "+" mean? Thanks! Zhiqiang Quoting "Jennifer Jackson" <jen@bx.psu.edu>:
Hi Zhiqiang,
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 attatched .png. Also listed here:
Click on "Add new Match Reads" Match Reads by: "Sequence Content" Sequence Match Type: "Regular Expression", using
^.{9}A.+
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:
^.{9}[^A].+
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 is mostly likely your second best choice (and more complicated). Tools in "Interval Operation" group will be of help if you go down that path.
Best, Jen Galaxy team
On 11/26/12 10:47 AM, Zhiqiang Shu wrote:
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!
Best, Zhiqiang
___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:
http://lists.bx.psu.edu/listinfo/galaxy-dev
To manage your subscriptions to this and other Galaxy lists, please use the interface at:
-- Jennifer Jackson http://galaxyproject.org
-------------------------- Zhiqiang Shu/Deng Lab Department of Biological Science Florida State University 319 Stadium Dr. Tallahassee, FL, USA, 32306-4295 zshu@bio.fsu.edu
Hi Zhiqiang, The regular expressions I shared are just filters. It strictly either finds sequences with an A at base position 10 (first expression) or without an A at base position 10 (second expression). All starting left -> right along the sequence text (or string). This may or may not be enough to do what you need. I just wanted to give this as an option in case it would, or perhaps it is a starting place if you can tune it to be appropriate. If your data contains more than just these two populations, or will not split appropriately based on this criteria, then this method will most likely not be the one you want to use. The plus "+" sign means "one or more" of the proceeding variable, which in this case was a dot "." that matches any single character with the exception of a newline (end of line) character, generally noted by a dollar sign "$". Regular expressions can be simple or quite tricky - a short crib is on the form help for the the tool "Filter and Sort -> Select", or you can search the web for many more comprehensive guidelines, as there is a component of artistry to creating these. Experimenting and testing to see what works is the best advice, when used. Good luck! Jen Galaxy team On 11/27/12 10:07 AM, Zhiqiang Shu wrote:
Thank you so much, Jennifer!
Your method is pretty good for filtering the sequences that have "A" at the 10th base position.But I'm sure if it have employed 10 bp complementary criterion? For the antisense, the 5' will be T (in complementary to A). Besides, my library contains other small RNAs. That's why I concern output files of sense and antisense must have 10bp complementary.
Another quick question, in you regular expression, what does "+" mean?
Thanks!
Zhiqiang
Quoting "Jennifer Jackson" <jen@bx.psu.edu>:
Hi Zhiqiang,
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 attatched .png. Also listed here:
Click on "Add new Match Reads" Match Reads by: "Sequence Content" Sequence Match Type: "Regular Expression", using
^.{9}A.+
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:
^.{9}[^A].+
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 is mostly likely your second best choice (and more complicated). Tools in "Interval Operation" group will be of help if you go down that path.
Best, Jen Galaxy team
On 11/26/12 10:47 AM, Zhiqiang Shu wrote:
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!
Best, Zhiqiang
___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:
http://lists.bx.psu.edu/listinfo/galaxy-dev
To manage your subscriptions to this and other Galaxy lists, please use the interface at:
-- Jennifer Jackson http://galaxyproject.org
-------------------------- Zhiqiang Shu/Deng Lab Department of Biological Science Florida State University 319 Stadium Dr. Tallahassee, FL, USA, 32306-4295 zshu@bio.fsu.edu
-- Jennifer Jackson http://galaxyproject.org
participants (4)
-
Everson,Richard -
Jennifer Jackson -
Peter Cock -
Zhiqiang Shu