title: sequencing data analysis of C. albicans CHS1 gene by Taq. amplification
design: (1)DNA Sample Detection. The detection of DNA samples mainly includes two methods: Agarose Gel Electrophoresis: This method analyzes the degree of DNA degradation and checks for RNA and protein contamination; Nanodrop: This technique measures the purity of DNA by calculating the OD 260/280 ratio.(2) DNA Quantification using Qubit.Qubit is used for precise quantification of DNA concentration. Samples with a concentration of over 0.5 g are used for library construction.(3) Library Construction.3.Once the DNA samples pass quality control, they undergo end repair and an A-tail addition. Sequencing adapters are ligated to both ends of the fragments, followed by purification. No PCR amplification is performed during this step, resulting in a PCR-free library. (4). Library Quality Control.After library construction is complete, an initial quantification is performed using Qubit 2.0. Subsequently, the insert size of the library is assessed using an Agilent 2100. Once the insert size meets expectations, qPCR is utilized to accurately quantify the effective concentration of the library (3 nM) to ensure quality. (5). Sequencing.After passing quality checks, libraries are sequenced on the Illumina platform based on the effective concentration and data output requirements. (6). Data Assembly.For samples that require demultiplexing, the process begins with separating the samples based on their barcodes to obtain the raw data for each sample, followed by the removal of barcodes and primers. The R1 and R2 sequence data are then assembled using FLASH software to generate tags. For samples that do not require demultiplexing, the R1 and R2 sequence data are directly assembled using FLASH software to create tags.