Usually bacteriophage contains modified bases, but we don't know what are those modified bases. In above situation how can i go about sequencing such virus.
In any NGS method can help, you no needs to know the sequences.
Thank You...Abdul-Aleem Mohammad.
The thing is it has many modified bases, i don't have an idea what modified bases they are. Bacterio -phages are very small, do u think its a good idea to go for NGS? . i wanted to know what is the chemistry or method used to sequence the modified bases.
Do u mean the base itself modified the chemistry? Or the mutations?
Base modifications..... like C-5-methylcytosine (m5C), N4-methylcytosine (m4C), N6-methyladenine (m6A), 5-hydroxymethylcytosine (hm5C) and derivatives of it with sugar residues attached (ghm5C), and 5-hydroxymethyluracil (hm5U).
Thank you very much....David Farringdon Spencer.
"Phage T4 of course does encode its own DNA polymerase and that may be a critical clue". Good point. Even i was thinking that what may be the difference between other DNA polymerase and polymerase of the phage? We have many new technologies for sequencing but i don't know that we can use those to sequence and say about bacteriophage's genome and its modification with exact place. Thank u.
Thank you...........
I will go through the T4 DNA polymerase. I used to think how page DNA escapes from the bacterial nuclease, what kind of modifications? in which place we have modified bases?
If i want to target any bacteriophage using chemically modified antisense oligo-nuceotides i need correct sequence. Still i have many questions in my mind, your answer and some clue helped me a lot . Thank you sir.
I successfully sequenced two phages (Delftia phage PhiW-14; and Bacillus phage SP-15) both of which have hypermodified bases replacing about 50% of the thymine residues. The problem with most next-generation sequencing technologies is that they CAN sequence through modifications and the scientist may not recognize the fact that the genome is modified. PacBio sequencing indicates the presence of modifications but, except for simple methylations, not the identity. The latter must be analyzed enzymologically and chemically (see: http://www.ncbi.nlm.nih.gov/pubmed/19082548)
Thank you .................Andrew Kropinski.
Ya in these kind of situations, its not about just sequencing but about correct sequencing.
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