Nextera XT fragment size and MiSeq v3 read length clashing ?

[BioGenomics]

Hi all,

I am planning a bacterial genome sequencing project (5 Mb) for around 80 samples to get good coverage. As the input material is limited, I need to use the Nextera XT kit (or standard Nextera kit might work too if we get 50 ng). However, I also want to use the MiSeq v3 kit for the better quality and longer 2 X 300bp reads (25 M reads). Nextera XT size distribution of 300 bp means I'll run into the flowcell and waste read length.

Did anybody use this combination already (Nextera XT + Miseq v3 PE 300 bp) and did you perform a size selection step for frags 500 bp and higher ? Any other suggestion ?

thanks a lot !

Greg

[nucacidhunter]

After PCR you can clean up with 0.5x bead as suggested by protocol which will remove most of fragments below 500 bp, unless you want some overlap between read 1 and read 2.

[BioGenomics]

After PCR you can clean up with 0.5x bead as suggested by protocol which will remove most of fragments below 500 bp, unless you want some overlap between read 1 and read 2.

Thanks for this. Well some overlap would be nice indeed, as this might improve assembly later on. I indeed read this trick, hope this also works as well with SeraMag beads (the ones I use). I also heard that the Transposomes/DNA ratio could be modulated to increase fragment size (lower Transpo conc), but may be more tricky... the bead trick seems safer (and repeatable).

[nucacidhunter]

Well some overlap would be nice indeed, as this might improve assembly later on.

I think decision on overlap and its length should be made prior to library prep in consultation with bioinformatician who is going to do assembly.

hope this also works as well with SeraMag beads

It should work with SeraMag home brew as well. Its cut off with different ratios can be determined by titration using sheared DNA (DNA ladder is a poor choice for titration).

I also heard that the Transposomes/DNA ratio could be modulated to increase fragment size (lower Transpo conc), but may be more tricky... the bead trick seems safer (and repeatable).

Modifying Transposon/DNA ratio will affect the fragment average size, but still there will be smaller fragments which can be easily removed using beads. If you want to increase size, using more DNA is better for yield than reducing transposon amount.