Genome gaps

[Al'Thor]

Hi all,

I am using the MiSeq platform (nextera XT, v2 paired-end chemistry, 500 cycles) for resequencing of bacteria to a well defined reference genome.

My question - assuming the prep is good, 20-40x coverage and there are good Phred scores what is the likelyhood of assembly gaps remaining that can't be fixed informatically and thus would need additional sequencing?

Cheers
Al'Thor

[Brian Bushnell]

Probably near 100% chance of at least one assembly gap; Illumina coverage is fairly erratic, especially with extreme or variable GC content. You might be able to minimize variability by using a PCR-free protocol and gathering the DNA when the cells are not in an exponential growth phase.

Also, the assembly quality depends on the repeat structure of the genome, which presumably is not known.