More mixed reads....help urgent

[sanju0891]

Hello,

i have been lately facing problems with my junior run. i have more mixed reads in my run which is like 50%. i performed 3 runs, cpb is 1 and all have enrichemnt above 20% (~35%).

In my last run i have reduced my cpb to half (0.5cpb) but it is still the same.

previously it used to work perefctly fine but recently all the problems are showing up. i use kapa qPCR for quantification.

onemore issue is during the bead enrichemnt process...washes with the enhancing buffer does not yield the white DNA pellets like it used to before...literally sometimes no pellet after 2 washes.... Maybe a problem with the kits?

i used the lot nos 93926520 and 93931420...... anyone encountering problems with these lot numbers.

[GenoMax]

I hope you are already talking with Roche tech support if you suspect the kits.

[sanju0891]

yes i am....i have sent all my data and LOT numbers of all my kits and waiting for their reply

[prakitnavin]

I think that the problem may be cause in library preparation step. Can i ask you some question?
What 's application did you perform? Short gun or Amplicon sequencing?
If you perform Amplicon sequencing, How long does your template?

Mixed read may be cause by the short fragment presented in your library. You should remove the short-fragment and checked by using bioanalyzer before perform a next run.