Hello, all
I am still working on the DEG analysis of miRNA.
I have successfully trimmed out the adapter and index sequences and mapped them using bowtie2.
Then, I have used samtools to convert sam file to bam file.. and finally got the .bam files.
Here, I mapped our fastq file to mouse whole genome reference file instead of mirBase hairpin. fa file..
(mapping rate is around 90%) (Note I have 6 samples including 3 controls)
For DEG analysis of miRNA( differentally expressed miRNA), I am trying to use cufflink-cuffdiff pipeline.
By accident, I did not put the -g genes.gtf option.
cufflinks -p 4 -g genes.gtf -o xxx_clout xxx.bam
when, I put the -g genes.gtf option (since I want to annotate our result), my genes.fpkm_tracking file have 25000 genes with annotation.
When, I put the command without -g genes.gtf option, my genes.fpkm.tracking file has 1829 lines. (annotation.. e.g. CUFF.1 CUFF.2 ..... CUFF.1829)
Could you please someone let me know why these two files are so different even the different is only -g genes.gtf option?
Thanks
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illinois.ks
With the -g option, you'll get results for every entry in the gtf file plus any novel entries. Without it, you'll only get information for entries that cufflinks assembles.
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Devon Ryan
Thank you!
Then, for the miRNA annotation, do I need to use -g option?
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illinois.ks
It's unlikely to hurt and might increase the quality of the results a bit, at least if the GTF file is decent.
Thank you Devon,
BTW, I got some weird result. Before I got your answer, I have run two analyses with -g option for cufflinks and without -g option for cufflinks.
Based on the description, the results should not have much difference.
BUT, the DEG results were different. Could you please give some comments?
I understand the little difference for fpkm values for each samples however, the status of significant is also different. Please see the result.
It means I will have different DEGs.. How can I handle this?
This is the part of output of gene_exp.diff file (from cuffdiff) with -g option for cufflinks.
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 Lypla1 chr1:4807892-4846735 C A OK 66.3389 96.7347 0.544179 1.44694 0.0102 0.0350251 yes
XLOC_000002 XLOC_000002 Tcea1 chr1:4857693-4897909 C A OK 20.174 32.1997 0.674552 2.0687 0.0005 0.00330423 yes
XLOC_000003 XLOC_000003 Atp6v1h chr1:5083172-5162549 C A OK 46.3804 46.7117 0.0102699 0.0315399 0.9549 0.971887 no
XLOC_000004 XLOC_000004 Oprk1 chr1:5588492-5606133 C A NOTEST 0 0 0 0 1 1 no
XLOC_000005 XLOC_000005 Rb1cc1 chr1:6214661-6276104 C A OK 4.88699 8.12632 0.733656 2.20416 5e-05 0.000499381 yes
..
This is the part of output of gene_exp.diff file (from cuffdiff) without -g option for cufflinks.
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 Lypla1 chr1:4807892-4846735 C A OK 69.8295 88.1472 0.336078 0.906617 0.1208 0.245285 no
XLOC_000002 XLOC_000002 Tcea1 chr1:4857693-4897909 C A OK 21.6891 28.7428 0.406232 1.2759 0.0293 0.0890071 no
XLOC_000003 XLOC_000003 Atp6v1h chr1:5083172-5162549 C A OK 48.015 43.1173 -0.155218 -0.484344 0.39715 0.564343 no
XLOC_000004 XLOC_000004 Oprk1 chr1:5588492-5606133 C A NOTEST 0 0 0 0 1 1 no
XLOC_000005 XLOC_000005 Rb1cc1 chr1:6209234-6276104 C A OK 5.58876 7.63383 0.449879 1.38708 0.0185 0.0634292 no
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