Almost all reads are nonperfect guide matches

[stupi...@gmail.com]

Hi all,

 I recently ran the count spacer.py on my amplified library and go tthe following:

Number of perfect guide matches: 3884
Number of nonperfect guide matches: 28011797
Number of reads where key was not found: 1251157
Number of reads processed: 29266838
Percentage of guides that matched perfectly: 0.0
Percentage of undetected guides: 98.2
Skew ratio of top 10% to bottom 10%: Not enough perfect matches to determine skew ratio

My library is customized and cloned into lentipuroguide plasmid without the cas9. I wonder what could be wrong here since the key can be found. I tried to adjust the key region start but cannot solve the issue. Also, some of my guides are only 18 nucleotide long. i wonder if this will affect the run using count spacer?

Thank you very much for your help. 

Regards,

Shi Hao

[brian.ia...@gmail.com]

You need to change this line as well, 20 to 18.

guide = read_sequence[start_index:(start_index + 20)]