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  • qoiopipq
    Junior Member
    • Apr 2016
    • 7

    Tissue specific 3' UTRs using DEXSeq

    I used IsoSCM to identify alternative 3' UTRs with different lengths in different tissue types(output files are GTFs containing tissue specific 3' UTRs), then I want to quantify the differential usage of these alternative 3' UTRs across different tissue types. I know that neither Ensembl nor UCSC GTF reference file contains the tissue specific 3' UTRs, so I tried to re-annotate IsoSCM output GTF files with reference GTF. The idea is that I want to get a GTF file containing all tissue specific 3' UTRs, which can be used for the following dexseq_count step.

    But, if multiple 3' UTRs/terminal exons (same start site, different stop sites) are annotated with same transcript ID, dexseq_prepare_annotation will only include the longest one in the GFF output.
    Here is an example of two possible 3' UTRs of gene Fubp1 in thymus GTF file:
    Code:
    chr3	sol	exon	152232395	152233253	.	+	.	gene_id "Fubp1"; gene_name "Fubp1"; p_id "P24872"; transcript_id "NM_057172"; tss_id "TSS5999";
    chr3	sol	exon	152232395	152232485	.	+	.	gene_id "Fubp1"; gene_name "Fubp1"; p_id "P24872"; transcript_id "NM_057172"; tss_id "TSS5999";
    Two different 3' UTRs of gene Fubp1 in liver GTF file:
    Code:
    chr3	sol	exon	152232395	152233965	.	+	.	gene_id "Fubp1"; gene_name "Fubp1"; p_id "P24872"; transcript_id "NM_057172"; tss_id "TSS5999";
    chr3	sol	exon	152232395	152233885	.	+	.	gene_id "Fubp1"; gene_name "Fubp1"; p_id "P24872"; transcript_id "NM_057172"; tss_id "TSS5999";
    One more 3' UTR of gene Fubp1 in spleen GTF file:
    Code:
    chr3	sol	exon	152232395	152232532	.	+	.	gene_id "Fubp1"; gene_name "Fubp1"; p_id "P24872"; transcript_id "NM_057172"; tss_id "TSS5999";
    After merging them and put through dexseq_prepare_annotation step, I only got one exon to represent 3' UTR:
    Code:
    chr3	dexseq_prepare_annotation.py	exonic_part	152232395	152233965	.	+	.	transcripts "NM_057172"; exonic_part_number "018"; gene_id "Fubp1"
    So, I am wondering how can I include all alternative 3' UTRs in the GFF file, then I can use it to count for the usage of these alternative 3' UTRs ??

    Cheers

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  • GATTACAT
    Reply to Nine Things a Sample Prep Scientist Thinks About Before Sequencing
    by GATTACAT
    Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
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  • SEQadmin2
    Nine Things a Sample Prep Scientist Thinks About Before Sequencing
    by SEQadmin2


    I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.

    Here are nine questions we think about, in roughly the order they matter, before...
    06-18-2026, 07:11 AM

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