Entering edit mode
9.4 years ago
512788522
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20
After running TopHat, I hava run the following command directly(because of no replicates for each condition, i.e normal versus mutation):
cuffdiff -p 8 \
-o ./cuffdiff_out \
-L normal,mut \
-u ./genes.gtf \
./tophat/accepted_hits.bam \
./tophat_mut/accepted_hits.bam
In addition, I also have performed the differential expression analysis in the following way:
cuffmerge -g ./genes.gtf \
-p 8 \
-o ./cuffmerge_out \
-s ./ucsc.hg19.fa assembly.txt
cuffdiff -p 8 \
-o ./diff_out \
-L normal,mut \
-u ./cuffmerge_out/merged.gtf \
./tophat/accepted_hits.bam \
./tophat_mut/accepted_hits.bam
There was great difference in their output. In the first way, the gene_exp.diff
looked like:
test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant
A1BG A1BG A1BG chr19:58858171-58874214 normal mut OK 33.5662 14.8501 -1.17654 -0.611634 0.50845 0.867476 no
A1BG-AS1 A1BG-AS1 A1BG-AS1 chr19:58858171-58874214 normal mut OK 1.17516 1.66153 0.499655 0.071299 0.84635 0.94951 no
A1CF A1CF A1CF chr10:52559168-52645435 normal mut NOTEST 0 0 0 0 11 no
but in the second way, it looked like:
test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant
XLOC_000001 XLOC_000001 DDX11L1 chr1:11873-29370 normal mut NOTEST 0 0 00 1 1 no
XLOC_000002 XLOC_000002 OR4F5 chr1:69090-70008 normal mut NOTEST 0 0 00 1 1 no
XLOC_000003 XLOC_000003 LOC100132062,LOC100133331 chr1:323891-328581 normal mut NOTEST 0 0 0 0 1 1 no
The test_id
and gene_id
looked so strange, and the number of differentially expressed genes detected were 16 and 51,respectively. Why was there the great difference? What did cause the bias in the final results?
Thanks!