forward and reverse sequance

asankaf · 02-04-2009, 10:29 PM

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

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

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...

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02-04-2009, 10:29 PM	#1
asankaf Member Location: clayton Join Date: Jan 2009 Posts: 10	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?

05-26-2009, 07:36 AM	#2
Melissa Senior Member Location: Switzerland Join Date: Aug 2008 Posts: 124	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?

05-26-2009, 12:18 PM	#4
sklages Senior Member Location: Berlin, DE Join Date: May 2008 Posts: 628	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

05-27-2009, 08:48 AM	#6
Pepe Member Location: Germany Join Date: Mar 2009 Posts: 28	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...