Cycles for NGS

I'm following the nature protocol paper, trying to use gecko2 library.

In the paper for the NGS it is suggested to use 80 cycles, but do you think that 75 cycles will be enough?

I will use the NextSeq.

Many thanks


Login or Signup to leave a comment
Fuchsia Kelpiealmost 3 years ago

We have been running 80 cycles of Read 1 and 8 cycles of Index 1 on the NextSeq using a 75-cycle kit.

Mint Balorabout 2 years ago

In your Nature paper, you suggested more than 100 reads per sgRNA after plasmid extraction in NGS. I have 4 libraries, each has 100,000 sgRNAs. So I need 400M reads. To be able to have these many reads, I need a NovaSeq6000 lane which is not available for us currently. The other option is to request for 2 MiSeq runs. In Miseq, I only can have 50 reads per sgRNA. in your opinion, do you think 50 reads per sgRNA is OK?

Fuchsia Kelpieabout 2 years ago

For 4 libraries of 100k guides, you will need 40M reads. You can try running a MiSeq for 50 reads/sgRNA and analyze the data to see if you need another MiSeq run. If your library is not too skewed, 50 reads/sgRNA should be sufficient for QC.

Mint Balorabout 2 years ago

Thanks!

Find your community. Ask questions. Science is better when we troubleshoot together.
Find your community. Ask questions. Science is better when we troubleshoot together.

Have a question?

Contact support@scifind.net or check out our support page.