stringTie Warning message

Question

[blooming.daisy333]

im using strinTie as assembler for my project. and have got following warning messages. can anyone kindly help me to explain and resolve this issue ???

[05/18 14:00:48] Loading reference annotation (guides)..
GFF warning: merging adjacent/overlapping segments of Cotton_A_18708_BGI-A2_v1.0 on chr1 (28601099-28601311, 28601314-28601385)
GFF warning: merging adjacent/overlapping segments of Cotton_A_32496_BGI-A2_v1.0 on chr10 (65978961-65979005, 65979010-65979033)
GFF warning: merging adjacent/overlapping segments of Cotton_A_27269_BGI-A2_v1.0 on chr8 (54954026-54954250, 54954253-54954306)
GFF warning: merging adjacent/overlapping segments of Cotton_A_27269_BGI-A2_v1.0 on chr8 (54954026-54954306, 54954309-54954387)

the command line used is as follows:

./stringtie /data/memona/sorted.bam -v -p 60 -G /data/memona/genome.gff3 -C -A -o /data/memona/hisat_assembled_transcript2.gtf

thank you so much

[Tm]

You are using GFF annotation file while running strintie, but it is always better to use GTF file. Stringtie has issued a warning as there are overlapping segments in your GFF file.

Here, GFF warning: "merging adjacent/overlapping segments of Cotton_A_18708_BGI-A2_v1.0 on chr1 (28601099-28601311, 28601314-28601385)" means segment "28601314-28601385" is overlapping with segment "28601099-28601311" of chr1. Thus, it is merging both of these segments and will consider it as one and proceed.

[blooming.daisy333]

thank you so much for the guidance. i have converted the GFF3 format to GTF and its giving the same warning.

is there any other possible solution???

would it effect the subsequent results as im interested to use it to determine Alternate splicing events???

[andrew.j.skelton73]

Have you looked at your reference GFF file in IGV at the coordinates listed?

[Tm]

Can you give more information like, are these segments representing same feature or different?

[blooming.daisy333]

what i have done is to align the RNA-seq data with Hisat2 and used that alignment to assemble the transcripts by stringtie and getting this warning. these two joined points are CDS of the same mRNA. the gene annotation file looks like this at these points:

chr1    GeneWise    mRNA    28601099    28601385    100 -   .   ID=Cotton_A_18708_BGI-A2_v1.0;Name=Cotton_A_18708;Shift=1;evid_id=evm.TU.supercontig_41.27-D14
chr1    GeneWise    CDS 28601314    28601385    .   -   0   Parent=Cotton_A_18708_BGI-A2_v1.0
chr1    GeneWise    CDS 28601099    28601311    .   -   0   Parent=Cotton_A_18708_BGI-A2_v1.0

kind help is need that would it effects the results????

Answer 1

[Tm]

1. Please check whether you are using the latest version of stringtie or not? Because latest version has better GFF parsing.
2. Also try using command below:

gffread -E annotation.gff -o- |less

Its more detail can be obtained here. So, check if you are getting any warnings or not.

1. If you don't mind using genome and annotation file from other sources like ensembl and NCBI, then please try using those gffs.

If still warnings are encountered, it is better to post it in stringtie github page and get proper clarity of the issue.

[blooming.daisy333]

thanks for the guidance. as per suggestion, i have run gffread and have not encountered with any warning. the ouput looks like this :

$ ./gffread -E /data/memona/annotatio                                                                                        n/arboreum.gff3 -o- | less
# ./gffread -E /data/memona/annotation/arboreum.gff3 -o-
# gffread v0.9.12
##gff-version 3
chr1    GLEAN   mRNA    84156   84764   1.00    -       .       ID=Cotton_A_10375_BGI-A2_v1.0
chr1    GLEAN   CDS     84156   84764   .       -       0       Parent=Cotton_A_10375_BGI-A2_v1.0
chr1    GLEAN   mRNA    94827   95120   0.90    +       .       ID=Cotton_A_10374_BGI-A2_v1.0
chr1    GLEAN   CDS     94827   95120   .       +       0       Parent=Cotton_A_10374_BGI-A2_v1.0
chr1    GLEAN   mRNA    303008  304950  0.68    -       .       ID=Cotton_A_10373_BGI-A2_v1.0
chr1    GLEAN   CDS     303008  303100  .       -       0       Parent=Cotton_A_10373_BGI-A2_v1.0
chr1    GLEAN   CDS     303853  303936  .       -       0       Parent=Cotton_A_10373_BGI-A2_v1.0
chr1    GLEAN   CDS     304122  304211  .       -       0       Parent=Cotton_A_10373_BGI-A2_v1.0
chr1    GLEAN   CDS     304332  304421  .       -       0       Parent=Cotton_A_10373_BGI-A2_v1.0
chr1    GLEAN   CDS     304738  304950  .       -       0       Parent=Cotton_A_10373_BGI-A2_v1.0

should i consider it OK or I shuld go for other parameters checking ???