Cufflinks - Assigning Transcripts Or Exons

Question

[AW]

I have mapped paired end reads to my reference genome using TopHat and then obtained FPKM values through Cufflinks. I ran cufflinks without a gtf file as I am doubtful about its quality.

I was wondering how transcripts and exons were assigned in the transcripts.gtf output? Without a gtf file how are exons grouped together into transcripts? (see below)

Additionally, these transcripts are present in the genes.fpkm_tracking file but without length or coverage information. What does this mean about these transcripts?

gi|321228270|ref|NW_003456354.1|    Cufflinks    transcript    47231    73196    808    -    .    gene_id "CUFF.7"; transcript_id "CUFF.7.1"; FPKM "10.0383916498"; frac "0.494670"; conf_lo "9.300440"; conf_hi "10.776343"; cov "33.488707";
gi|321228270|ref|NW_003456354.1|    Cufflinks    exon    47231    48414    808    -    .    gene_id "CUFF.7"; transcript_id "CUFF.7.1"; exon_number "1"; FPKM "10.0383916498"; frac "0.494670"; conf_lo "9.300440"; conf_hi "10.776343"; cov "33.488707";
gi|321228270|ref|NW_003456354.1|    Cufflinks    exon    48894    49044    808    -    .    gene_id "CUFF.7"; transcript_id "CUFF.7.1"; exon_number "2"; FPKM "10.0383916498"; frac "0.494670"; conf_lo "9.300440"; conf_hi "10.776343"; cov "33.488707";
gi|321228270|ref|NW_003456354.1|    Cufflinks    exon    49688    49882    808    -    .    gene_id "CUFF.7"; transcript_id "CUFF.7.1"; exon_number "3"; FPKM "10.0383916498"; frac "0.494670"; conf_lo "9.300440"; conf_hi "10.776343"; cov "33.488707";
gi|321228270|ref|NW_003456354.1|    Cufflinks    exon    50723    50852    808    -    .    gene_id "CUFF.7"; transcript_id "CUFF.7.1"; exon_number "4"; FPKM "10.0383916498"; frac "0.494670"; conf_lo "9.300440"; conf_hi "10.776343"; cov "33.488707";

Any help would be much appreciated,

Thanks

Answer 1

[Mikael Huss]

The first line represents a transcript, and the next four are exons (look at column 3). The fact that the entries have the same transcript_id attribute tells you that these exons belong to the transcript represented by the first line. Without a GTF file, Cufflinks tries to infer where the exons start and end and how to map exons to transcripts.

Answer 2

[JC]

First, Cufflinks without a GTF file works trying to infer the transcripts in the sample using a coverage search and local assembly, reads with splice evidence or information for paired-ends reads are used to infer the exons.

The information for length and coverage for each transcript is wrote in the isoforms.fpkm_tracking file.