I am attempting to sequence Gram negative bacteria (Enterobacteriacea) strains using the MiSeq platform. I extracted the DNA using automated Magnapure system and I tried Qiagen manual Kit method, I check the purity ratio using nanodrop. I am making DNA libraries using Illuminas Nextera kit which involves the use of a transposome to fragment the DNA.
After the fragmentation I carry out the PCR with the indexes and the PCR clean-up. I then run the sample on a Fragment analyser to assess the fragmentation but the sizes are peaking around 1000-1500 bp. The problem is that if the fragments are greater then 1000bp and run on the MiSeq they will not cluster correctly and an accurate sequence can't be produced.
We're inputting DNA at 0.2ng/uL (Qubit) and I tried to put less input and extend the fragmentation time from 5 to 7 minutes but it didn't seem to have any effect.
I was wondering is anybody else doing similar work ? Have they come across similar problems of the enzyme underfragmenting ? Any help would be appreciated
Are you size selecting after Nextera . (similar to Ampure beads)?
Anukana Bhattacharjee Thank you for your reaction!
yes after the tagmentaion and the PCR I make a cleaninig selection step with Ampure beads: PCR product 50 ul on which I add 30 ul beads then make 2 washing ethanol 80%, resuspension in 52 ul RSB. At this point when doing the QC using the fragment analyser I frequently get big fragment. Then normalization (using the kit) it is beads based.
You can check this blog
https://www.keatslab.org/blog/affectofheatingtimeondnafragmentation
https://www.keatslab.org/blog/ampureandnot-so-simple
Do some optimization. Good luck!
- Badges
- Science method