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cufflinks result interpretation

i have 2 rna sample and when did cufflinks with aligned bam files , i have got result for sample1 in transcript.gtf file -

2       Cufflinks       transcript      29643815        29738975        1       +       .       gene_id "ENSMUSG00000039844"; transcript_id "ENSMUST00000238899"; FPKM "0.0325281086"; frac "0.000455"; conf_lo "0.012566"; conf_hi "0.052490"; cov "1.448822";
2       Cufflinks       exon    29643815        29643914        1       +       .       gene_id "ENSMUSG00000039844"; transcript_id "ENSMUST00000238899"; exon_number "1"; FPKM "0.0325281086"; frac "0.000455"; conf_lo "0.012566"; conf_hi "0.052490"; cov "1.448822";
2       Cufflinks       exon    29679133

for sample2 - 

2       Cufflinks       transcript      29643815        29738975        163     +       .       gene_id "ENSMUSG00000039844"; transcript_id "ENSMUST00000238899"; FPKM "1.0183970793"; frac "0.071810"; conf_lo "0.851414"; conf_hi "1.185380"; cov "14.202789";
2       Cufflinks       exon    29643815        29643914        163     +       .       gene_id "ENSMUSG00000039844"; transcript_id "ENSMUST00000238899"; exon_number "1"; FPKM "1.0183970793"; frac "0.071810"; conf_lo "0.851414"; conf_hi "1.185380"; cov "14.202789";

both sample have same exon numbers but rpkm value is diffrenent . what interpretation can we get from this result. what is meaning of this result?

rna-seq cufflinks alignment result interpretation
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updated by Kristoffer Vitting-Seerup Kristoffer Vitting-Seerup • written by priyadhawan11234 • priyadhawan11234

2 answers

You mean FPKM, not RPKM. The interpretation is that the expression is higher in sample 2 when compared to sample 1. That is all.

Cufflinks is out dated, by the way. You should be using HISAT2 / StringTie if your aim is to perform transciptome assembly. Otherwise, a pseudoaligner like Salmon or Kallisto should suffice.

Also, FPKM and RPKM units should not be used if your end-goal is to perform differential expression analysis.

Kevin

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written by Kevin Blighe Kevin Blighe

Grammar nazi here: Salmon in its current version now performs selective alignment :)

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written by ATpoint ATpoint

what-else interpretation can we do from above output ? for example can we know transcript length and exon numbers in both samples from this output?

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written by priyadhawan11234 • priyadhawan11234

That information is right there in your output. Please take another look.

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written by Kevin Blighe Kevin Blighe

I had thought that, generally, HISAT2 and StringTie were the upgrades to TopHat2 and Cufflinks, respectively... or am I wrong?

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written by Kevin Blighe Kevin Blighe

Please note that if you want to compare the expression of the two samples you need to first run Cuffmerge and then requantify the samples on the joint transcriptome. You can read more at cufflinks website or here.

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written by Kristoffer Vitting-Seerup Kristoffer Vitting-Seerup

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