Low number of reads from Miseq runs

[Otternonsense13]

Hi all,

I'm trying to perform guide-seq protocol (https://www.protocols.io/view/guide-...xygxmwool8j/v1) using 293T cells.
The library prep steps are intuitive: gDNA extraction, fragmentation, end-repair, TA adapter ligation, PCR1, and PCR2. The thing that puzzled me is that I always got very few reads from my experiment (2K-200K reads) from the Mi-seq run. Tapestation for PCR2 product showed a good size range of library DNA (tho the average length of library is ~1kb, a bit big; see pic). So far, I ruled out the possibility of incomplete denaturation (my colleague's samples are pooled together with mine in the same run, he got good amount of reads). What else could be contributing to this? Thank you!

[Ben3]

Hello there,

1) Are you and your colleague using the same library kit? If not, that could cause an issue. Whenever you pool different library prep kits it's very difficult to evenly pool together both libraries. They have different sizes and slightly different chemistries that always make it too difficult to make things even. I suggest pooling only libraries made with the same prep whenever you're able.

2) If you are both using the same library kit, what are the sizes of your colleague's library? If they are smaller than your library then they will get more reads if everything was pooled evening. Shorter libraries preferentially bind to the flow cell. This also applies to small fragments, so hopefully your colleague's samples were clean.

3) If you used the same library prep kit and you had similar sizes, then there's a slight possibility that they were not pooled evening. Can you briefly explain how you pooled your samples?

Feel free to include any other information about combining their preps and I can give additional suggestions.