I'm interested in sequencing a region in a bacterial genome that has high variance. This region is an integration site for various mobile genetic elements. When I PCR this region, I can see amplicons of 300 bp to 11 kbp are present simultaneously. Are there are any sequencing technologies that anyone knows of/can suggest that would best suit this situation? So far, I've been reading into the Illumina targeted and custom genotyping services, PacBio targeted sequencing (>20 kB), and Oxford Nanopore MinIon sequencing. Any suggestions would be greatly appreciated!
Thank you so much for your response, Tobias! It's always nice to get a response from a friend :).
Hi Nadia Morson
I agree with tobias answers with long read NGS possibility, but why not get a step froward and zoom out the sequence to analyze invariable sequences and design longPCR? Using this longPCR as template you could thereafter use specific primers. But it depends manly on your questions and money you can/want to invest.
fred
Hi, it does not sound that your variable region has a repetitive character. Then, to me, it does not make any difference whether you use PacBio or Illumina. Both technologies are suitable for full-genome sequencing - and with both technologies you will be able to assemble the region of interest (provided coverage is sufficient).
However, that's quite some money and depends on how many strains you wish to compare. You may rather think of an enrichment procedure, such as PCR. Can you clone the PCR fragments, incl. those of 11 kb size? When you say that you see several amplicons at the same time, is this due to mispriming?
Best, Ralf
Some Suggestions: use statistical methods ， study the vast amounts of data that sequencing machines churn out ， find features that are unique to different species, and then use those features ，help assemble the right sequences
I have the analytical experience in this field, If you are willing to provide some data, I can generate some inspiration, Get some methods, And tell you what I found my email: [email protected]
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