Dear researchers, greetings to all of you
There is a situation that is not in my field of study but that I need to understand for an experiment. I isolated lysogenic phage, performed many experiments (host range, MOI, adsorbtion curve etc.). I have the genome of the phage, I want to sequence its genome and also show its place in the host bacteria genome. However, I am encountering a problem.
Sequencing is performed using Nanopore, but a complete (whole) genome sequence cannot be obtained. When I run the genome on the agarose gel, I see that it is a whole and large genome. But we can't get results from sequencing. This is not the case in lytic phage genomes.
Does anyone have any suggestions regarding this? What should be considered in sequencing the lysogenic phage genome? Has anyone experienced something like this before?
Thank you a lot
Not sure I can provide any help, but can you provide more information? What is the host? What is the approximate genome size?
And would a brute force approach be possible by just sequencing the lysogen instead of the phage?
If you're having trouble sequencing the genome of a lysogenic phage using Nanopore sequencing, there are a few things you can try. First, you might consider using alternative sequencing technologies such as Illumina or PacBio. Alternatively, you could try increasing the coverage depth or using a hybrid sequencing approach. If none of these work, you could try assembling the genome in contigs or using additional bioinformatic tools like long-read assemblers or hybrid assemblers.
Hello, can you please tell me what library prep kit are you using for phage sequencing on ONT? One factor could be the high modifications in phage genomes that make sequencing difficult. For this, PCR-based library preps could be more successful, as they would remove the modifications.
Best regards and good luck.
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