Hello everyone,
These days I've read many papers regarding RNA-seq and its corresponding analysis, I found when it comes to assembly part, almost every paper would tell how many contigs and scaffolds, which are basically called 'unigenes', have formed out of cleaning reads.
Since I'm not familiar with the assembly process I'm looking on the internet and find a pic elucidating the procedure involved which I've attached below. As in the pic, there are four contigs which comprise two scaffolds, name them as 1,2,3 and 4.
MY QUESTION is first why it cannot be the case that contig 1,2,3 or 4 itself rather than larger sequence (scaffolds) they combined would be recognized as 'unigene'.
Second question would be how to determine which two contigs in the pic comprising scaffolds which represent the real transcriptomes, since basically contig 2 and 3 are also able to be gathered as a scaffold, all of them have certain gaps inbetween.
Many thanks in advance.
The picture you provide isn't the best to represent the difference between contigs and scaffolds. In short:
- you start assembly from short NGS reads
- overlapping parts of the reads are joined to form longer sequences, called the contigs
- For the contigs what you don't know is that if you have 3 contigs, what is their order? Which one presents the beginning of ie. a chromosome, which one is in the middle and which one at the end? Another thing that you don't know is which strand the contig represents? Contig 1 might be on the + strand, contig 2 on the negative strand. Another unknown is that the contig might be inverted, so it's important if we try to match its sequence when reading it from left to right or from right to left
You did not include the whole image. The original image (found by searching Google Image) shows that some contigs can be combined based on paired read information, giving two scaffolds. Contigs 2 and 3 cannot be "gathered" as scaffolds because there is no paired-read information justifying their assembly into a scaffold. By the way, this is not a very good image for describing the process because it does not actually show how the paired-reads are connected.
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