Data Presentation

 

We have constructed a sgRNA library that targets the entire human genome, containing 63,950 sgRNA sequences. After the library construction, we conducted Sanger and NGS sequencing to detect the error rate, coverage, and uniformity of the library.

 

 

Low Error Rate

 

A batch of 30 clones was randomly selected for Sanger sequencing after completion of library construction. The sequencing results showed that 100% of the sgRNA sequences in the randomly selected clones matched the theoretical sequence completely.

 

Figure1. Sanger Sequencing results

 

 

High coverage, uniform distribution, high stability between batches

 

The coverage and distribution uniformity of the library are important indicators for evaluating the quality of the library. We used NGS to verify the coverage and stability between batches. Coverage refers to the proportion of actually detected sequences to designed target sequences included in the library. Ideally, different sgRNA sequences are evenly distributed in the library. However, due to inevitable bias introduction during the PCR and cloning processes, some sgRNA reads are relatively low, while others are relatively high. Therefore, uniformity is used to evaluate the distribution uniformity of sequences in the library. The distribution skew ratio is commonly used as a metric, with values <10 considered as a qualified library. The smaller the value, the better the uniformity.

 

 

We have built four batches of the library described above, and the NGS detection analysis results are as follows:

 

Batch number	Diversity of designed library	Actual diversity of the library	Coverage	Skew ratio
01	63950	63950	100%	2.24
02	63950	63940	99.98%	2.39
03	63950	63937	99.98%	2.91
04	63950	63943	99.99%	2.29