Cuffdiff splicing.diff output

Question

[zhengyuguobuaa]

Dear all,

I run cufflinks/cuffmege/cuffdiff pipeline on my data. But I have some problem on how to interpret the splicing.diff results. Here are some of my results:

test_id    gene_id    gene    locus    sample_1    sample_2    status    value_1    value_2    sqrt(JS)    test_stat    p_value    q_value    significant
TSS2038    XLOC_001278    Q91YR5    chr1:164462249-164478706    g1    g2    OK    0    0    0.358701    2.93163e-09    4.59102e-10    1.52192e-06    yes
TSS745    XLOC_000478    NP_001074727    chr1:138362954-138428088    g1    g2    OK    0    0    0.372363    6.93969e-07    1.27085e-07    0.000210643    yes
TSS30120    XLOC_020929    Q80TA1,uc008wvi.1    chr5:30559157-30849030    g1    g2    OK    0    0    0.278089    3.08277e-06    5.95185e-07    0.00049326    yes

I am wondering how can I find out the splice sites from the splicing.diff results. I'd like to check the significant splicing events visually on a genome browser. But The locus provided in splicing.diff seems to be the locus of the gene/transcript, from which I can't locate the position where the splicing event happens.

Also, I am also confused about how to figure out the AS isoforms that are compared by cuffdiff. For example, if a genes has 5 splicing variant and cuffdiff perform a spilcing test on this gene. How could I identify the two isoforms that are compared by cuffdiff?

Thanks a lot!

Answer 1

[Kristoffer Vitting-Seerup]

Unfortunately cufflinks does not analyse the actual splicing pattern (it "just" tests for differences in expression of transcripts originating from the same promoter).

You can however use the R package spliceR (Bioconductor page, article) which is made for analysing splicing patterns from RNA-seq data, and have an easy wrapper for importing all the relevant data from CuffDiff (just take a look at the vignette).

If you want to visualise the differences spliceR can furthermore generate a GTF file where the transcripts are coloured by their Isoform Fraction (IF) values (defined as percent of gene expression originating from a transcript), which can be uploaded to directly to UCSC genome browser (see example in article).

Hope this helps.

[mfahim]

Hi Kristoffer,

I want to use SpliceR but am not sure how to rebuild my cummeRbund library. Can you please post instructions (commands) on how to set up the library for SpliceR?. I am working with Arabidopsis.

Thank you.

Fahim