Hi! I could amplified one gene (4kb), but it was divided in 3 fragments (size: 2.9kb, 2.7kb and 0.8). When I tried to alignment this 3 fragments to get the complete structure, they told me that is not possible because of the bias over ratio. Somebody now about this (bias over ratio). Thank you
Hi,
Firstly, could you clarify the sizes of the gene and plasmid (assuming the fragment sizes you gave include plasmid fragments) - also, do you know what digestion was used?
Could I ask what technology you're using for this process and whether it's DNA or RNA you're sequencing?
The process for sequence alignment and assembly shouldn't be difficult depending on which technology / process you're using - most of the modern protocols do this automatically with NGS technology...though I think they work on bigger sizes than 4kb (I might be wrong)
For s small sequence of 4kb, assuming you know what gene you're working with, you should be able to piece together the fragments based on a reference sequence from NCBI or other databases...if you know which plasmid was used and where the RE sites are - you can use that to piece your fragments together as well...
For a de novo genes / sequences, you won't have a reference to work from - it's a little more complicated...I found a site that explains the process quite nicely :)
http://www.yourgenome.org/facts/how-do-you-put-a-genome-back-together-after-sequencing
It boils down to how much information you have on the gene / sequence. More information makes it easier to piece together - less information will mean more time and more work...don't make more work for yourself if you don't need to :) So if you know what gene it is - use the known sequence data to help you...
In regards to the "bias" - it really depends on what technology you're working with and how accurate the reads are...Illumina is currently the most widely used technology, but there are others...
Not sure if that helps - but I do think it is possible regardless of the bias in play - especially if you have a reference gene to work from...take a look at PCR Assembly protocols - that might also help :)
Your fragments should be in overlapping manner,so after aligning join them correctly after cutting the extras.
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