Annotation of miRNA from NGS

Question

[illinois.ks]

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

Answer 1

[Devon Ryan]

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.

[illinois.ks]

Thank you!

Then, for the miRNA annotation, do I need to use -g option?

[Devon Ryan]

It's unlikely to hurt and might increase the quality of the results a bit, at least if the GTF file is decent.

[illinois.ks]

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