[Retracted] High-Throughput Screen of Natural Compounds and Biomarkers for NSCLC Treatment by Differential Expression and Weighted Gene Coexpression Network Analysis (WGCNA)

Table 2

Detailed information of RNA-seq analysis pipeline.

Step	Analyses	Software	Script	Input	Main output
1	Prepare raw data	Trimmomatic ver.0.36	trimmomatic-0.36.jar PE -threads 12 -phred33 -trimlog xx.log xx.fq1 xx.fq2 -baseout	FASTQ file	FASTQ file
2	Quality control	FastQC	fastqc -t 10 -f fastq -o out xx.fq	FASTQ file from step 1	FASTQ file, multiqc_report.html
3	Alignment	HISAT	hisat2 -p 20 -x hg38.fa -1 xx_clean_1P.fq.gz -2 xx_clean_2P.fq.gz -S XX.sam	FASTQ file from step 2	Sam file
4	Sort	Samtools	samtools sort -@ 20 -O bam -o xx.bam xx.sam	Sam file from step 3	Bam file
5	Transcript assembly	StringTie	stringtie -e -p 30 -G hg38.gff -o xx.gtf xx.bam	Bam file from step 4	gtf file
6	Merged transcripts	StringTie-- merge	stringtie --merge -o merged.gtf gtflist	All gtf file from step 5	gtf file
7	Reads count	StringTie--eB	stringtie -B -e -p 30 -G merged.gtf -o xx.gtf xx.bam	gtf file from step 6 and Bam file from step 4	Gallgown input file and gtf file
8	Generated gene expression (FPKM)	Ballgown	gene_expression = gexpr(bg)	Output files from StringTie--eB	Gene expression tables
9	Differential expression analysis	DESeq2 Wrapper of TBtools	Selection criteria of DEGs: and	Gene expression tables	Volcano plot and differential gene list
10	KEGG pathway analysis	R package clusterProfiler and http://org.Hs.eg.dbdata set	Parameter: , ,	Differential gene list	KEGG pathway tables
11	Gene expression visualization	TBtools	Default parameters	Expression of top genes	Heatmaps
12	General analysis of WGCNA	“WGCNA” R package	Power of (scale free )	FPKM table	Plots and forms of module connectivity
13	Selecting module and hub genes	“WGCNA” R package	and	Output files from WGCNA	Module and gene list
14	Network	Cytoscape	Default parameters; The criteria for edge filter: weight and weight	Output files from WGCNA	Network plots

The table lists analysis steps, software, and main scripts in our pipeline. Starting from the input FASTQ files produced by sequencing and finally generating the results of candidate medicine and genes for NSCLC cancer research.