Differential gene expression Normal lymph nodes vs Metastatic lymph nodes

Differential expressionwas assessed for each gene using an exact test analogous to Fisher's exact test butadapted for overdispersed data. TMM normalization and Tagwise dispersion (individual dispersion for each gene) were used to adjust for abundance differences across samples, and DEGs were extracted. Genes were considered differentially expressed only at a P-value <0.05 and at False Discovery Rate (FDR) < 0.05. Genes with log2-fold changes of more than 2 or less than -2 were then selected to ensure that only robust changes were considered.Differentially expressed genes between two groups were annotated using HUGO Gene Nomenclature Committee^TM(HGNC) (https://www.genenames.org).

针对每个基因，采用类似费希尔精确检验（Fisher's exact test）但适配过度分散数据的精确检验方法，进行差异表达分析。通过TMM归一化与基因特异性离散度（即每个基因各自的离散度）校正不同样本间的表达丰度差异，并提取差异表达基因（DEGs）。仅当P值小于0.05且错误发现率（False Discovery Rate, FDR）小于0.05时，方可将基因判定为差异表达基因。随后筛选log₂倍变化大于2或小于-2的基因，以确保仅纳入变化幅度可靠且显著的差异表达基因。采用人类基因命名委员会（HUGO Gene Nomenclature Committee^TM，HGNC）对两组间的差异表达基因进行注释，注释网址为https://www.genenames.org。