gravatar for wangmingcheng1992

40 minutes ago by

China_LanZhou_lzu

All samples should be used to assembly one result, my workflow:
1: Trinity --seqType fq --max_memory 20G --samples_file sample_file.config --genome_guided_bam ref_sorted.bam --genome_guided_max_intron 10000 --CPU 60

Find assembled transcripts as: 'trinity_out_dir/Trinity-GG.fasta''

"sample_file.config" is a txt file; suppose have 4 samples and everyone has 3 replicates, "sample_file.config" like below:
sample1 sample1-rep1 sample1-rep1_1.fq sample1-rep1_2.fq
sample1 sample1-rep2 sample1-rep2_1.fq sample1-rep2_2.fq
sample1 sample1-rep3 sample1-rep3_1.fq sample1-rep3_2.fq
sample2 sample2-rep1 sample2-rep1_1.fq sample2-rep1_2.fq
sample2 sample2-rep2 sample2-rep2_1.fq sample2-rep2_2.fq
sample2 sample2-rep3 sample2-rep3_1.fq sample2-rep3_2.fq
sample3 sample3-rep1 sample3-rep1_1.fq sample3-rep1_2.fq
sample3 sample3-rep2 sample3-rep2_1.fq sample3-rep2_2.fq
sample3 sample3-rep3 sample3-rep3_1.fq sample3-rep3_2.fq
sample4 sample4-rep1 sample4-rep1_1.fq sample4-rep1_2.fq
sample4 sample4-rep2 sample4-rep2_1.fq sample4-rep2_2.fq
sample4 sample4-rep3 sample4-rep3_1.fq sample4-rep3_2.fq

before run trinity assembly, align all the fq files to the elephant shark geome(hisat), then merge(samtools) all the sam format result to "ref_sorted.bam";
2: Transcript Quantification with salmon
trinityrnaseq-v2.11.0/util/align_and_estimate_abundance.pl --transcripts ./trinity_out_dir/Trinity-GG.fasta --seqType fq --samples_file sample_file.config --output_dir salmon_transcript_quantification --aln_method bowtie2 --thread_count 60 --est_method salmon --trinity_mode --prep_reference
3: DE analyse with DESeq2
trinityrnaseq-v2.11.0/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix salmon_transcript_quantification/salmon.gene.counts.matrix --method DESeq2 --samples_file sample_file.config --contrasts contrasts.file --output Differential_Expression_Analysis

"contrasts.file" like below(suppose sample1 vs sample2, sample3 vs sample4 for DE analyse):
sample1 sample2
sample3 sample4

reference: github.com/trinityrnaseq/trinityrnaseq/wiki



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