Hi, 

We would like to know how can we enhance the barcoded reads for our CRISPR Gecko v2 library sequencing? 

When we sequenced our Geckov2 PCR2 samples, we found that there is only about 2% barcoded reads among the samples (8.6 million reads). Also, the reads range from 0-~70, and the 50th percentile is 2 reads. 

Any explanation for this problem? We think that there is a ligation problem during the PCR steps since when we sequenced our libraries (before infecting the cells), the number of barcoded reads was 86% in 1 million reads.

In addition, the extracted PCR2 band was about 350bp, which is supposed to be the right size. However, during the quality control process before the sequencing, there was another peak appeared at about 500bp. We do not know the reason. 

Thanks,

Reham