（已装RSEM、bowtie2）

  $ /util/align_and_estimate_abundance.pl --transcripts trinity.fasta --seqType fq --left 1.fq --right 2.fq --est_method RSEM --aln_method bowtie --trinity_mode --prep_reference --output_dir rsem1_outdir --thread_count 48    (Estimating Transcript Abundance)

              (note: RSEM requires bowtie)

              --trinity_mode will automatically generate the gene_trans_map and use it

              --prep reference (builds target index)

              --output_dir <string>  write all files to output directory

                         If Paired-end:   --left <string>

                                                  --right <string>                                                 

                         or Single-end:  --single <string>

                         or  --samples_file <string> tab-delimited text file indicating biological replicate relationships.
                                                 cond_A cond_A_rep1 A_rep1_left.fq A_rep1_right.fq
                                                 cond_A cond_A_rep2 A_rep2_left.fq A_rep2_right.fq
                                                 cond_B cond_B_rep1 B_rep1_left.fq B_rep1_right.fq
                                                 cond_B cond_B_rep2 B_rep2_left.fq B_rep2_right.fq
                                          if single-end instead of paired-end, then leave the 4th column above empty.

 

  $ /util/abundance_estimates_to_matrix.pl --est_method RSEM sampleA.results sampleB.results  --gene_trans_map none --name_sample_by_basedir     (Build Transcript and Gene Expression Matrices)

                         --name_sample_by_basedir requires that you have all your expression results organized into separate directories

                         The 'counts.matrix' file is used for downstream analyses of differential expression. The TMM.EXPR.matrix file is used as the gene expression matrix in most other analyses.

                         例子：/util/abundance_estimates_to_matrix.pl --est_method kallisto --gene_trans_map Trinity.fasta.gene_trans_map --out_prefix kallisto --name_sample_by_basedir sampleA/abundance.tsv sampleB/abundance.tsv

  $ R

   > source("https://bioconductor.org/biocLite.R")

   > biocLite("edgeR")

   > biocLite("limma")

   > biocLite("DESeq2")

   > biocLite("ctc")

   > biocLite("Biobase")

   > install.packages("gplots")

   > install.packages("ape")

 $ /Analysis/DifferentialExpression/run_DE_analysis.pl --matrix genes.counts.matrix --method edgeR --samples_file samples_described.txt   (Differential Expression Analysis)

            建立samples_described.txt

                    cond_A sampleA
                    cond_A sampleB
                    cond_B sampleC
                    cond_B sampleD

  $ /Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix TMM.EXPR.matrix -P 1e-3 -C 3   (Extracting and clustering differentially expressed transcripts)