Abi prism bigdye sequencing kits

The HBV EnhII/BCP/PC region was amplified in the subjects with a successfully detected HBV genotype using nested PCR13 (link). The primers for the first and second round of the nested PCR are listed in Supplementary Table S1. Direct DNA sequencing was carried out by using ABI PRISM BigDye sequencing kits and an ABI 3730 Genetic Analyser (Applied Biosystems, Foster City, CA). The HBV sequences were aligned and analysed using MEGA 5.0 software. After the alignment, the nucleotide with the highest frequency at each site in the HBV EnhII/BCP/PC region from the HBV persistent carriers was termed as the wild type nucleotide. Nucleotide substitutions with the other three nucleotides and deletions at each site were termed as mutations. A site with combined mutation frequencies >10% from all the participants was termed a hotspot.

EnhII/BCP/PC region was amplified by nested‐PCR. The primers of the nested‐PCR were listed in our previous study.15 The amplified products were then purified and sequenced by using ABI PRISM BigDye sequencing kits and an ABI 3730 Genetic Analyser (Applied Biosystems, CA). The sequences were aligned and analyzed using MEGA 6.0 software. Wild type at each site was defined as the nucleotide with the highest frequency among the HBV persistent carriers. Nucleotide substitutions with the other three nucleotides or indels were defined as mutations. A site with combined SNV frequencies >10% was defined as a hotspot.

The exon regions of the BRCA1 and BRCA2 genes were amplified by 31 pair and 41 pair primers, respectively (Supplementary Tables 1 and 2), using the SureCycler 8800 (Agilent, Penang, Malaysia). Direct DNA Sanger sequencing was carried out by ABI PRISM BigDye Sequencing Kits and ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The obtained sequences of BRCA1 and BRCA2 were aligned and analyzed using MEGA 5.0 software compared with GenBank accession numbers NM_007294.3 and NM_000059.3, respectively. We observed 48 variants in the discovery stage and selected candidate variants in validation stage with following criterions: (1) frameshift, nonsense, and missense variants; (2) minor allele frequency (MAF) less than 1% according to the Asian samples from 1000 Genomes Project [14 (link)]; and (3) the clinical classification identified by ENIGMA [15 (link)] was ascertained from BRCA exchange (https://brcaexchange.org/) ranging from Class 3 to Class 5. Then, we performed subsequent variant screening on the MassARRAY System (Agena Bioscience, San Diego, CA) among multicancers and the corresponding controls. For quality control, positive controls of candidate variants were used in each chip. Each recurrent variant identified from screening was doubly validated by Sanger sequencing.