SEQanswers

SEQanswers (http://seqanswers.com/forums/index.php)
-   General (http://seqanswers.com/forums/forumdisplay.php?f=16)
-   -   forward and reverse sequance (http://seqanswers.com/forums/showthread.php?t=1149)

asankaf 02-04-2009 10:29 PM

forward and reverse sequance
 
in most of the mutation detection software tools we need to provide forward and reversce trace files along with the reference sequence files.
What is this forward/reverse trace files and why we use this kind of aproach?

Melissa 05-26-2009 07:36 AM

That's the Sanger's way to detect SNPs. All Sanger sequencing requires the forward and reverse primer of the amplicon. The forward sequences are sequenced from the forward primer and vice versa. Therefore, both forward and reverse sequences should reverse complement each other.

Say, you want to detect SNPs in individual 1 & 2. You have forward and reverse sequences from both individuals (shown below). At first look, it seems like there's a T/C SNPs. But, there's a high probability that base C in sequence 2F is a sequencing error because it's not present in the reverse strand.

1F - AATAA
2R - AATAA
2F - AACAA
2R - AATAA

SOLiD can detect the orientation of the reads. I was told that this is one of the advantage of SOLiD over other platforms. I really don't see the importance. Any thoughts?

swbarnes2 05-26-2009 10:29 AM

Quote:

Originally Posted by Melissa (Post 5459)
SOLiD can detect the orientation of the reads. I was told that this is one of the advantage of SOLiD over other platforms. I really don't see the importance. Any thoughts?

I think it's helpful in RNA-seq, or maybe small RNAs?

It doesn't matter much for DNA sequencing.

sklages 05-26-2009 12:18 PM

SOLiD can distinguish between a simple sequencing error and a SNP, as there must be at least two changes in colorspace to change one base in basespace. That's the advantage of SOLiD.

cheers,
Sven

Melissa 05-26-2009 08:41 PM

Quote:

Originally Posted by swbarnes2 (Post 5462)
I think it's helpful in RNA-seq, or maybe small RNAs?

It doesn't matter much for DNA sequencing.

I was informed that it'll be helpful in RNA-seq too. Two genes that are located next to each other can be distinguished if they are transcribed in different direction. Quite an unique situation. Don't think it will be useful for most people.

Pepe 05-27-2009 08:48 AM

Having orientation does not seem very important because most experiments don't have the ability to see it. I bet there are many interesting surprises once it becomes a standard.
For example, I've heard people doing RNA-seq experiments and finding reads in reverse orientation that align to introns from some genes. No clue what they are there for...


All times are GMT -8. The time now is 06:41 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.