Problem while using Tophat 2.1.0 to search gene fusions

Question

[Pallondyle]

I'm using Tophat 2.1.0 tophat-fusion-post to search for gene fusion. The tutor told me to download human_genetic, other_genetic, and nt* data from https://ftp.ncbi.nlm.nih.gov/blast/db/ . However, I did not see the first two in it. Which files I need to download in current version of Tophat?

[Pallondyle]

It seems that I can run this without blast direction existing, but alternative splicing cannot be found this way.

tophat-2.1.0.Linux_x86_64/tophat-fusion-post -p 20 --num-fusion-reads 1 --num-fusion-pairs 2 --num-fusion-both 5 index/bowtie1/hg38_bowtie

[Mon Sep 23 12:17:18 2024] Beginning TopHat-Fusion post-processing run (v2.1.0)
-----------------------------------------------
[Mon Sep 23 12:17:18 2024] Extracting 23-mer around fusions and mapping them using Bowtie
[Mon Sep 23 12:17:22 2024] Filtering fusions
        0 fusions are output in ./tophatfusion_out/potential_fusion.txt
[Mon Sep 23 12:17:24 2024] Blasting 50-mers around fusions
[Mon Sep 23 12:17:24 2024] Generating read distributions around fusions
[Mon Sep 23 12:17:24 2024] Reporting final fusion candidates in html format
        num of fusions: 0
-----------------------------------------------
[Mon Sep 23 12:17:24 2024] Run complete [00:00:05 elapsed]

Answer 1

[GenoMax]

However, I did not see the first two in it.

This is because NCBI changed versions of the blast database to v.5 a few years back. tophat (being an old program) was using the older v.4 blast databases. You can still find archival versions of v.4 databases from https://ftp.ncbi.nih.gov/blast/db/v4/ . Note: These are from the year 2020 and are not being updated. If you are OK with that download all of your databases from link above. You can't mix and match v.4 and 5 databases.

If you are looking for fusions you would be best served by looking for an alternate program instead of tophat..

[Pallondyle]

I run the program again and I received following errors:

BLAST Database error: No alias or index file found for nucleotide database [blast/human_genomic] in search path [/data/lastry::]
BLAST Database error: No alias or index file found for nucleotide database [blast/nt] in search path [/data/lastry::]

I then received a list of fusions but the quantity exceeded expectations:

1       ENSA    chr1    150629446       LINC02301       chr14   82692985        1       27      1       0.00
1       PRSS23  chr11   86821763        MOB4    chr2    197516146       3       2       2       0.00
1       PABPC1  chr8    100706958       PABPC3  chr13   25097504        4       169     2       0.00
1       FOXA1   chr14   37592329        TTC6    chr14   37715505        4       16      34      0.00
1       PAPOLA  chr14   96502599        AK7     chr14   96437834        3       2       5       0.00
1       AC099850.1      chr17   59107590        VMP1    chr17   59838294        4       4       5       0.00
1       LRP1B   chr2    141480394       PLXDC1  chr17   39109389        2       3       2       0.00
1       BCAS4   chr20   50795172        BCAS3   chr17   61353587        9       2       12      0.00
1       BCAS4   chr20   50795172        BCAS3   chr17   61368326        103     112     193     0.00
1       SEPTIN9 chr17   77482143        SEPTIN9-DT      chr17_GL383566v1_alt    72446   1       5       1       0.00
1       SMARCA4 chr19   10986592        CARM1   chr19   10904950        2       3       1       0.00
1       TEX101  chr19   43401035        ARL8B   chr3    5180807 3       3       3       0.00
1       ARFGEF2 chr20   48922009        SULF2   chr20   47736941        21      12      32      0.00
1       SULF2   chr20   47786404        ZNF217  chr20   53593755        15      5       28      0.00
1       SRPK2   chr7    105168057       NKAIN3  chr8    62863649        1       7       0       0.00

It seems that the tophat is really unreliable. I decide to change my software.

[Pallondyle]

Can you please give me some software suggestions?

[dsull]

STAR-Fusion or Arriba for fusion gene detection.

[Pallondyle]

Thx very much