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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? |
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? |
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It doesn't matter much for DNA sequencing. |
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 |
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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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