Frame issues

[komalsrathi]

Hi,

I have Arriba fusion caller results (run with Gencode v27 equivalent to Ensembl 90) and wanted to test the following out-of-frame fusion:

#gene1	gene2	strand1(gene/fusion)	strand2(gene/fusion)	breakpoint1	breakpoint2	site1	site2	type	direction1	direction2	split_reads1	split_reads2	discordant_mates	coverage1	coverage2	confidence	closest_genomic_breakpoint1	closest_genomic_breakpoint2	filters	fusion_transcript	reading_frame	peptide_sequence	read_identifiers	sample_name
CHMP1A	DPEP1	-/-	+/-	16:89651569	16:89625844	splice-site	intron	deletion/read-through/5'-5'	upstream	downstream	7	6	1	118	36	high	.	.	duplicates(4),low_entropy(1)	GCTTGGTCGGTTCGATCGCCGCCGGGACCTGACACCGCCCGGAGTTGGCGTCCCTTCTCCCTCTCCGAGTGCTGCTCCTGTCATTGTGGCCATGGACG___ATACCCTGTTCCAGTTGAAG___TTCACGGCGAAGCAGCTGGAGAAGCTGGCCAAGAAGGCGGAGAAGGACTCCAAGGCGGAGCAGGCCAAAGTGAAGAAG|TCTCTTCGTCTGTCAAGTAGAAGTCATGGATGTACCTACTTTACGACAGTCCTGCTGTGAGCACAAAGGTACCAGCCACTCAGATCACCCTCGGATCAAG___CTCTGTCTCCCAGAACCACACAGAAGCCCCATCCACAGCCAACATGCAGACGCCACCGTGGCCATTTCGGATGATACCACTTCTGCACAGGCAGCCACAGGCAATGCGGACCTACCCACGCCCCCCACACACAGATCATCGCAG	out-of-frame	MDDTLFQLKFTAKQLEKLAKKAEKDSKAEQAKVKK|slrlssrshgctyfttvll*	.	0062396f-103a-4c25-bfdc-a746782768d9

I ran agfusion like this:

agfusion annotate \
--gene5prime CHMP1A \
--gene3prime DPEP1 \
--junction5prime 89651569 \
--junction3prime 89625844 \
-db agfusion.homo_sapiens.90.db \
-o CHMP1A-DPEP1

I got quite the opposite result from agfusion, predicting it to be an in-frame fusion:

$ cat CHMP1A-DPEP1.fusion_transcripts.txt
5'_gene,3'_gene,5'_transcript,3'_transcript,5'_strand,3'_strand,5'_transcript_biotype,3'_transcript_biotype,Fusion_effect,Protein_length,Protein_weight_(kD)
CHMP1A,DPEP1,ENST00000397901,ENST00000393092,-,+,protein_coding,protein_coding,in-frame,446,49.6940567

I am not sure why that is happening, any ideas/suggestions would be much appreciated.

[murphycj]

Thanks for bringing up this issue. I will take a look at this later tonight/tomorrow!

[komalsrathi]

Hi @murphycj

Did you get a chance to go over this? Thank you!

[murphycj]

Sorry for the belated reply. I was on vacation.

I've been looking at your case, and I see two issues here:

1. According to Ensembl annotation, the junction for DPEP1 never lands within the coding region. It usually lands between two exon that encode portions of the 5'UTR. So this should definitely not be called in-frame. I'll have to fix this.
2. There seems to be some kind of annotation difference between Ensembl and Gencode. Here are the transcript annotations I used for both genes on Ensembl:

CHMP1A
DPEP1

I used the coordinates and nearby sequences you provided for your fusion to manually compare with what's on Ensembl:

Gene	Relative exon number	Your results (Gencode)	Ensembl
CHMP1A	Exon -3	GCTTGGTCGGTTCGATCGCCGCCGGGACCTGACACCGCCCGGAGTTGGCGTCCCTTCTCCCTCTCCGAGTGCTGCTCCTGTCATTGTGGCCATGGACG	GGCGACCCCGGAAGTCCCCGCCGGGTGCAGCTTGGTCGGTTCGATCGCCGCCGGGACCTGACACCGCCCGGAGTTGGCGTCCCTTCTCCCTCTCCGAGTGCTGCTCCTGTCATTGTGGCCATGGACG
CHMP1A	Exon -2	ATACCCTGTTCCAGTTGAAG	ATACCCTGTTCCAGTTGAAG
CHMP1A	Exon -1	TTCACGGCGAAGCAGCTGGAGAAGCTGGCCAAGAAGGCGGAGAAGGACTCCAAGGCGGAGCAGGCCAAAGTGAAGAAG	TTCACGGCGAAGCAGCTGGAGAAGCTGGCCAAGAAGGCGGAGAAGGACTCCAAGGCGGAGCAGGCCAAAGTGAAGAAG
DPEP1	Exon +1	TCTCTTCGTCTGTCAAGTAGAAGTCATGGATGTACCTACTTTACGACAGTCCTGCTGTGAGCACAAAGGTACCAGCCACTCAGATCACCCTCGGATCAAG	GCAGAGTGGCTCCTCACAGCCTGAAGCTCATCCTTCTGCACGGGCCAGCCAGGCCAGCACAGAGGCACCAGGGCAGCAGTGCACACAGGTCCCCGGGGACCCCACCATGTGGAGCGGATGGTGGCTGTGGCCCCTTGTGGCCGTCTGCACTGCAGACTTCTTTCGGGACGAGGCAGAGAGGATCATGAGGGACTCCCCTGTCATTGATGG
DPEP1	Exon +2	CTCTGTCTCCCAGAACCACACAGAAGCCCCATCCACAGCCAACATGCAGACGCCACCGTGGCCATTTCGGATGATACCACTTCTGCACAGGCAGCCACAGGCAATGCGGACCTACCCACGCCCCCCACACACAGATCATCGCAG	GCACAATGACCTCCCCTGGCAGCTGCTGGATATGTTCAACAACCGGCTGCAGGACGAGAGGGCCAACCTGACCACCTTGGCCGGCACACACACCAACATCCCCAAGCTGAGGGCCGGCTTTGTGGGAGGCCAG

As you can see the CHMP1A portion that I pulled from Ensembl matches what you got, but the DPEP1 portion does not. According to your fusion, the junction position for DPEP1 is 89625844, which lands somewhere in the intron between the first two exons. Hence, the DEPEP1 portion of this fusion will include the full exon 2 and onward in the cDNA.

Let me know if I am misunderstanding something.

[komalsrathi]

@murphycj I think definitely 1 is an issue. For 2, are you looking at Ensembl 90?