Different genomes, different libraries

[Katerina Cvikova]

Hi everybody,

do you have a some experience to sequence different genomes prepare with different kit in one run on MiSeq?

We are planning to sequence about 40 BAC clones prepared by TruSeq PCR-Free, each BAC has 110 kbp, the one genome with size 120 Mbp and one genome with 3 Gbp, both prepared with Nextera DNA kit.

I am not sure how to pooled this library (molarity) to have similar coverage for each sample. Two bigger genomes should have lower coverage.

Any Idea?
Thanks

[Etherella]

It's ok to pool different libraries, although you'll have to demultiplex separately if the libraries have different indexing. If you want to get more coverage for your libraries of larger size, then I'd advise pooling them in adequate proportions.

What concerns me more is sequencing 3Gbp genome on Miseq, the instrument is not purposed to sequence genomes of that size.

40 BAC clones of 110 kbp is ~4,5Mbp in total + 120 Mbp Genome is 124,5 MBp. That should be fine to sequence on a miseq, but you might have troubles geting enough coverage for your BAC clones since most of the flowcell will probably be occupied by the 120 MBp Genome. I'd advise pooling 40 BAC clones in equalmolar, then pooling the resulting library with 120 Mbp genome 1:25 (1 part 40 clones pooled library, 25 parts Mbp genome).