Multiplexing for pooled CRISPR screen sequencing

Question

[Tuấn Anh]

Hi all,

We have been doing single-indexed, single-end sequencing for genome-wide CRISPR screen in our lab for a while. However, using Illumina prep kit, we could only sequence up to 24 samples per run. As we are now planing to do more screens with a smaller library size, it would be more practical to increase the maximum number of samples per run. For that reason, I would like to ask.

1. If we keep doing single-end sequencing, could we add more barcodes to our existing library indices? If yes, is there anything we should be aware of when designing the new barcodes? Could you please suggest any resources for barcode design?

2. If we switch to paired-end sequencing with dual-index barcode, I am aware that the data of output files will increase. However, given the same library, would the number of reads sequenced per run be equivalent to that of single-end sequencing? How much more expensive is the paired-end sequencing compared to the single-end?

Many thanks.

Answer 1

[GenoMax]

If we switch to pair-end sequencing with dual-index barcode

You can use dual-indexing with single end reads. You don't need to do paired-end sequencing.

How much more expensive is the pair-end sequencing compared to the single-end?

Will depend on your provider but paired-end reads give you no additional information in this application.

[Tuấn Anh]

Thank you GenoMax!

So would we just prepare the dual-indexed library and ask the sequencing service to do single-end sequencing?