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How Can Cufflinks Assemble De Novo Transcripts With Many Exons When They Have 0 Fpkm

Hi, I have seen many studies where they assembled transcriptomes (RNA-Seq) using cufflinks and usually it finds many novel transcripts but surprisingly most of them have 0 FPKM expression levels. Has any one noticed this ?

A good ex:

chr5    Cufflinks    transcript    19702133    19761803    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19702133    19702839    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19704533    19704567    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "2"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19705319    19705437    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "3"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19706257    19706391    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "4"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19706708    19706805    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "5"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19708313    19708453    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "6"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19709434    19709586    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "7"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19710326    19710331    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "8"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
chr5    Cufflinks    exon    19712308    19712467    1    -    .    gene_id "XLOC_150252"; transcript_id "TCONS_00425102"; exon_number "9"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";

.........

rna-seq cufflinks fpkm
• 5,183 views • link
updated by Ram Ram • written by biorepine biorepine

I posted few lines as biostar has limit on word size.

• 0 views • link
written by biorepine biorepine

I have noticed this as well in my RNAseq data have have yet to find an explanation.

• 1 views • link
updated by Ram Ram • written by bmpbowen • bmpbowen

Which paper/dataset, and which versions of tophat and cufflinks was used for the above analyses? There were some bugs in various older builds leading to assignment of 0 FPKM in cufflinks and cuffdiff runs, not sure how many of them still persist in v2.2+.. see e.g.

If there are alternate isoforms for the genes in question, it could potentially also be linked to cufflinks consideration of multiple isoforms per gene and probabilistic assignment of reads?

Due to similar issues and the somewhat black box nature of cufflinks (and limited design matrix setups), I've turned to other tools for abundance estimates (eXpress) and differential expression analyses (DESeq2, limma).

• 1 views • link
updated by Ram Ram • written by David Fredman David Fredman

Cufflinks is used to assembly transcripts, the FPKM was used to filter the results. For this purpose, cufflinks is worth to try.

• 0 views • link
written by pengchy pengchy

1 answer

As far as I know, Cufflinks doesn't assemble transcriptomes; [edit: it does; I didn't know very far, apparently]. Something like Trinity would do that for them. Do you mind linking us to the aforementioned studies?

Also, the text output you provided shows several exons with 0 FPKM, but if there are some exons in the lines below (that are not shown in your clipped output) with nonzero FPKM / expression it would make sense to assemble a transcript from the combination of exons that are expressed. This would be something downstream to the analysis done using Cufflinks, though.

• 0 views • link
written by Isaac Joseph Isaac Joseph

First sentence on the Cufflinks web page begins: "Cufflinks assembles transcripts...". So yes, it does.

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written by Neilfws Neilfws

I think the disagreement stems from what the word "assemble" actually means. Cufflinks assembles transcripts from reads that have been aligned to a known reference genome whereas Trinity assembles transcripts from reads when the genome sequence is not known.

• 60 views • link
written by Istvan Albert Istvan Albert

@ijoseph: all exons have 0 fpkm. please read the question again carefully. Cufflinks is one of well known software that could assemble novel transcripts in de novo as cleverly mentioned by both of the users above. Here is one study for your reference.

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written by biorepine biorepine

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