An empty "merged.gtf" file generated after running cuffmerge, How to solve the problem?

Question

[vuductp]

cufflinks v2.2.1

I have

1) essembles.txt

C20_GAGTGG_R1.tophat.out/cufflink_out/transcripts.gtf
C7_CGTACG_R1.tophat.out/cufflink_out/transcripts.gtf
D01_GTGAAA_R1.tophat.out/cufflink_out/transcripts.gtf
D04_GTGGCC_R1.tophat.out/cufflink_out/transcripts.gtf
D05_GTCCGC_R1.tophat.out/cufflink_out/transcripts.gtf
D14_GTTTCG_R1.tophat.out/cufflink_out/transcripts.gtf
D15_CGTACG_R1.tophat.out/cufflink_out/transcripts.gtf
F1W_AGTCAA_R1_51.tophat.out/cufflink_out/transcripts.gtf
F2L_AGTTCC_R1_51.tophat.out/cufflink_out/transcripts.gtf
F5W_ATGTCA_R1_51.tophat.out/cufflink_out/transcripts.gtf
F8L_CCGTCC_R1_51.tophat.out/cufflink_out/transcripts.gtf
H7_ACTGAT_R1.tophat.out/cufflink_out/transcripts.gtf
H8_ATTCCT_R1.tophat.out/cufflink_out/transcripts.gtf
I1_GTCCGC_R1.tophat.out/cufflink_out/transcripts.gtf
I2_GTGAAA_R1.tophat.out/cufflink_out/transcripts.gtf
I5_GTGGCC_R1.tophat.out/cufflink_out/transcripts.gtf
I6_GTTTCG_R1.tophat.out/cufflink_out/transcripts.gtf
K11_TAGCTT_R1_51.tophat.out/cufflink_out/transcripts.gtf
K12_GGCTAC_R1_51.tophat.out/cufflink_out/transcripts.gtf
K7_ACTTGA_R1_51.tophat.out/cufflink_out/transcripts.gtf
L11_GATCAG_R1_51.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC04_327bp_ATCACG_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC05_317bp_TTAGGC_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC06_338bp_ACTTGA_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC07_339bp_GATCAG_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC08_347bp_TAGCTT_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC09_366bp_GGCTAC_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC10_351bp_GTGGCC_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC11_347bp_GTTTCG_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC16_348bp_CGTACG_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC17_336bp_GAGTGG_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC20_362bp_ACTGAT_L001_R1.tophat.out/cufflink_out/transcripts.gtf
LTS17_MC21_370bp_ATTCCT_L001_R1.tophat.out/cufflink_out/transcripts.gtf
M5_GTGGCC_R1_51.tophat.out/cufflink_out/transcripts.gtf
M8_CGTACG_R1_51.tophat.out/cufflink_out/transcripts.gtf
Y4L_trim.tophat.out/cufflink_out/transcripts.gtf
Y4W_trim.tophat.out/cufflink_out/transcripts.gtf

2) Betta_splendens_v1.1.fa (reference genome)

3) Betta_splendens_v1.1.gene.gtf (reference gene sequence)

I run cuffmerge as follow :

/bio/package/cufflinks/2.1.1/cuffmerge -s ../Betta_splendens_v1.1.fa.fai -g ../Betta_splendens_v1.1.gene.gtf assemble.txt

it run successfully and generated a "merged.gtf" file. However, the file is empty without any information.

Could anyone please give me a solution? I highly appreciated for your help.

Thanks and best regards

[Bastien Hervé]

Hello, the tuxedo pipeline has been updated since a while now, see A: cufflink issues in Tuxedo protocol

-s option needs the fasta sequence so .fa not .fa.fai

I suggest you to put the full path of your files in your assemble.txt

[vuductp]

Thank you very much for your quick reply. Could I ask 1) I obtain .fa.fai file from the database. So to have a .fa file I need to convert the fa.fai file into fa. file, I am correct? 2) Could you please tell me more detail about this suggestion "I suggest you to put the full path of your files in your assemble.txt"? I'm sorry, I am new to the analysis. Thanks and best regards

[Bastien Hervé]

Please use the add reply grey button on my comment to answer my comment, so the thread will be better organized

I obtain .fa.fai file from the database.

What is your 'database' ? The .fai is an index of the .fa file. For comparison the .fa is a book and the .fai is just the index part. So you can not convert your .fai to .fa. You have to find the reference file .fa

Could you please tell me more detail about this suggestion "I suggest you to put the full path of your files in your assemble.txt"

Everytime you run a software you are in what is called the current directory, it is where you are in your arborescence, for example in your terminal if your prompt is : /home/vuductp/Documents/Software, you are in your Sofware directory.

You have two ways to fill a path the relative path and the full path

The full path is the path from the root (unix is /home), wherever you are in your arborescence, the path stay correct (i.e : /home/vuductp/Documents/Software)

The relative path is the path from where you are in your arborescence (i.e : if you already are in your /home/vuductp/, the relative path to your Software directory is Documents/Software). BUT if you move from /home/vuductp/ the path will break and will no longer be correct.

So, in your case if you have all your *.tophat.out directory in your current directory it is OK, if not i strongly suggest you to complete all paths

[vuductp]

Dear Bastien Hervé, Thank you very much for your patient response. I will take time to work on these issues and come back soon. Best regards