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Rui:RNAseq analysis on HL099
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===Cuffdiff=== * I never get the original command (listed in cufflinks website) worked! Just as when I run cufflinks, I have to convert bam to sam before running, otherwise, it will show segmentation fault. The original command * Run Cuffdiff: Take the annotated transcripts for your genome (as GFF or GTF) and provide them to cuffdiff along with the BAM files from TopHat for each replicate: cuffdiff annotation.gtf mock_rep1.bam,mock_rep2.bam \ knockdown_rep1.bam,knockdown_rep2.bam Run with accepted_hits.bam from tophat * nohup cuffdiff ~/iGenome/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ../Indx1/tophat_out/accepted_hits.bam ../Indx2/tophat_out/accepted_hits.bam & * [1]+ Segmentation fault nohup cuffdiff ~/iGenome/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ../Indx1/tophat_out/accepted_hits.bam ../Indx2/tophat_out/accepted_hits.bam Run with sorted.acceptedHits.sam from removalClonalhits.pl * nohup cuffdiff ~/iGenome/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ../Indx1/tophat_out/sorted.acceptedHits.sam ../Indx2/tophat_out/sorted.acceptedHits.sam & * Error: this SAM file doesn't appear to be correctly sorted! current hit is at chrX:64289, last one was at chrM:16509 Cufflinks requires that if your file has SQ records in the SAM header that they appear in the same order as the chromosomes names in the alignments. If there are no SQ records in the header, or if the header is missing, the alignments must be sorted lexicographically by chromsome name and by position. Convert bam to sam with header, then cuffdiff * samtools view -h accepted_hits.bam > accepted_hits.sam * nohup cuffdiff ~/iGenome/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/HL099/Indx1/tophat_out/cuffcompare/accepted_hits.sam ~/HL099/Indx2/tophat_out/cuffcompare/accepted_hits.sam & * gene_exp.diff file has ~1700 genes with significant... repeat direct-cuffdiff on HL098 to compare [636 genes with significance as same as ones using tophat-cufflinks-cuffcompare-cuffdiff methods] * Why no NGN3????
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