Pfam IsoformSwitchAnalyzerR

Question

[SpliceAndScript]

Hy everyone, I am using IsoformSwitchAnalyzerR (ISAR) for my isoform switching study. One of the functions in this tool is that it takes Pfam output file and tells us which protein domains are present/absent in isoforms. I did Pfam analysis on my FASTA file on ubuntu and trying to load into results into ISAR for downstream analysis. but ISAR is showing parsing, file formatting issues and is unable to read it. I have downloaded the latest version of ISAR, ran Pfam twice but still the same issue. Raw Pfam file is in domtblout format and have 21 columns. ISAR accepts 16 column files. I have done that too. I am failing in doing analysis on the Pfam file. If anyone has ever worked with Pfam file or is familiar with ISAR, please reach out to help.

Answer 1

[Kevin Blighe]

Hey,

It seems like you're running into a parsing issue with the Pfam domtblout file in IsoformSwitchAnalyzeR (ISAR). The tool expects the standard HMMER domtblout format, typically with 22 columns (based on HMMER 3.x documentation), but variations in column counting or file formatting can cause errors, especially if headers or descriptions are mishandled.

First, ensure you're using the latest development version of ISAR, as updates often fix parsing bugs. Install it from GitHub:

if (!require("devtools")) install.packages("devtools")
devtools::install_github("kvittingseerup/IsoformSwitchAnalyzeR")

Restart your R session after installation.

If you're using a local HMMER installation (current version is 3.4 as of 2025), double-check your command: hmmscan --domtblout output.txt Pfam-A.hmm your_sequences.fasta. Open the file in a text editor to confirm it's space-delimited without extra lines or rich text artifacts.

As an alternative, try the Pfam web server (https://www.ebi.ac.uk/interpro/search/sequence/). Submit your FASTA, wait for the email, then copy only the results table into a plain .txt file (not Word). Supply this to analyzePFAM().

If problems persist, share a sample of your Pfam file header and your ISAR code for more specific help.

Kevin