RNA-SEQ ANALYSIS
1. Library preparation and Sequencing
From the RNA samples to the final data, each step, including sample test, library preparation, and sequencing, influences the quality of the data, and data quality directly impacts the analysis results. To guarantee the reliability of the data, quality control (QC) is performed at each step of the procedure. The workflow is as follows:
Firstly, ribosomal RNA was removed from total RNA, followed by ethanol precipitation. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. Following is the workflow of library construction:
The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.
2. Results
Data Quality Control: The “e” represents the sequence error rate and Qphred represents the base quality value, Qphred=-10log10(e). The relationship between sequencing error rate (e) and sequencing base quality value (Qphred) is shown in the table below:
The distribution of quality score is shown in Fig.1:
Summary of Sequencing Data Information