Could not locate a HISAT2 index corresponding to basename

Question

[altobialmunthir]

I'm using HISAT2 version 2.1.0 on a Windows 10 system to align RNA-seq reads. Despite having the required index files in the correct directory, I keep encountering an error stating that HISAT2 cannot locate the index files.

Details:

Index Files: The index files are present in the directory C:/Users/Almunthir/Desktop. Here are the files listed:

C:/Users/Almunthir/Desktop/genome.1.ht2
C:/Users/Almunthir/Desktop/genome.2.ht2
C:/Users/Almunthir/Desktop/genome.3.ht2
C:/Users/Almunthir/Desktop/genome.4.ht2
C:/Users/Almunthir/Desktop/genome.5.ht2
C:/Users/Almunthir/Desktop/genome.6.ht2
C:/Users/Almunthir/Desktop/genome.7.ht2
C:/Users/Almunthir/Desktop/genome.8.ht2

HISAT2 Command:

In Command Prompt, I attempted to run the following command after setting the environment variable HISAT2_INDEXES:

set HISAT2_INDEXES=C:\Users\Almunthir\Desktop
"C:\Users\Almunthir\Desktop\hisat2.1\hisat2-align-l.exe" -x "genome" -U "C:/Users/Almunthir/Desktop/SRR26264823_trimmed.fastq" -S "C:/Users/Almunthir/Desktop/SRR26264823_aligned.sam"
Error Message:
Could not locate a HISAT2 index corresponding to basename "C:/Users/Almunthir/Desktop/genome"
Error: Encountered internal HISAT2 exception (#1)
Command: /hisat2.1/HISAT2~1 -x C:/Users/Almunthir/Desktop/genome -U C:/Users/Almunthir/Desktop/SRR26264823_trimmed.fastq -S C:/Users/Almunthir/Desktop/SRR26264823_aligned.sam

R Script Attempt:

I also tried running the command in R with the following script:

# Ensure all required files are present
required_files <- c("genome.1.ht2", "genome.2.ht2", "genome.3.ht2", "genome.4.ht2", "genome.5.ht2", "genome.6.ht2", "genome.7.ht2", "genome.8.ht2")
index_files <- list.files("C:/Users/Almunthir/Desktop", pattern = "genome.*\\.ht2", full.names = TRUE)
all_files_present <- all(sapply(required_files, function(file) any(grepl(file, index_files))))

if (!all_files_present) {
    stop("Not all required index files are present.")
}

# Set paths with the full path
hisat2_path <- "C:/Users/Almunthir/Desktop/hisat2.1/hisat2-align-l.exe"
index_path <- "C:/Users/Almunthir/Desktop/genome"  # Base name of the index
input_fastq <- "C:/Users/Almunthir/Desktop/SRR26264823_trimmed.fastq"
output_sam <- "C:/Users/Almunthir/Desktop/SRR26264823_aligned.sam"

# Set the HISAT2_INDEXES environment variable
Sys.setenv(HISAT2_INDEXES = "C:/Users/Almunthir/Desktop")

# Construct the command arguments
args <- c("-x", shQuote(index_path), "-U", shQuote(input_fastq), "-S", shQuote(output_sam))

# Print the command to verify it
cmd <- paste(hisat2_path, paste(args, collapse=" "))
cat("Command to be executed:\n", cmd, "\n")

# Run the command using system2
result <- system2(hisat2_path, args, stdout = TRUE, stderr = TRUE)

# Print the result
cat("Result of the command:\n", result, "\n")
Error Message in R:
Could not locate a HISAT2 index corresponding to basename "C:/Users/Almunthir/Desktop/genome"
Error: Encountered internal HISAT2 exception (#1)
Command: /hisat2.1/HISAT2~1 -x C:/Users/Almunthir/Desktop/genome -U C:/Users/Almunthir/Desktop/SRR26264823_trimmed.fastq -S C:/Users/Almunthir/Desktop/SRR26264823_aligned.sam

Request:

Could anyone help me identify why HISAT2 is not recognizing the index files and suggest a solution? Any guidance on how to correctly set the environment or adjust the paths would be highly appreciated.

[Ram]

Please use the formatting bar (especially the code option) to present your post better. You can use backticks for inline code (`text` becomes text), or use one of (a) the option highlighted in the image below/ (b) fenced code blocks for multi-line code. Fenced code blocks are useful in syntax highlighting. If your code has long lines with a single command, break those lines into multiple lines with proper escape sequences so they're easier to read and still run when copy-pasted. I've done it for you this time.

[GenoMax]

Did you make the index yourself? Where is the original fasta reference used to make the index and what is the command that was used?.

[altobialmunthir]

No, I did not make the index myself. I downloaded the pre-built index files for GRCh38 from a reliable source. The original FASTA reference used to make the index is not with me as I directly used the pre-built index files available online. Here is the list of index files I have on my desktop:

C:/Users/Almunthir/Desktop/genome.1.ht2
C:/Users/Almunthir/Desktop/genome.2.ht2
C:/Users/Almunthir/Desktop/genome.3.ht2
C:/Users/Almunthir/Desktop/genome.4.ht2
C:/Users/Almunthir/Desktop/genome.5.ht2
C:/Users/Almunthir/Desktop/genome.6.ht2
C:/Users/Almunthir/Desktop/genome.7.ht2
C:/Users/Almunthir/Desktop/genome.8.ht2

Since I did not generate these indices myself, I do not have the exact command used to create them. However, I am trying to use these files with the following command in Windows:

set HISAT2_INDEXES=C:\Users\Almunthir\Desktop
"C:\Users\Almunthir\Desktop\hisat2.1\hisat2-align-l.exe" -x "genome" -U "C:/Users/Almunthir/Desktop/SRR26264823_trimmed.fastq" -S "C:/Users/Almunthir/Desktop/SRR26264823_aligned.sam"
And here is the command I am trying to run in R:
# Set paths with the full path
hisat2_path <- "C:/Users/Almunthir/Desktop/hisat2.1/hisat2-align-l.exe"
index_path <- "C:/Users/Almunthir/Desktop/genome"  # Base name of the index
input_fastq <- "C:/Users/Almunthir/Desktop/SRR26264823_trimmed.fastq"
output_sam <- "C:/Users/Almunthir/Desktop/SRR26264823_aligned.sam"

# Set the HISAT2_INDEXES environment variable
Sys.setenv(HISAT2_INDEXES = "C:/Users/Almunthir/Desktop")

# Construct the command arguments
args <- c("-x", shQuote(index_path), "-U", shQuote(input_fastq), "-S", shQuote(output_sam))

# Print the command to verify it
cmd <- paste(hisat2_path, paste(args, collapse=" "))
cat("Command to be executed:\n", cmd, "\n")

# Run the command using system2
result <- system2(hisat2_path, args, stdout = TRUE, stderr = TRUE)

# Print the result
cat("Result of the command:\n", result, "\n")
Despite having the index files in the specified directory, I still encounter the following error:
Could not locate a HISAT2 index corresponding to basename "C:/Users/Almunthir/Desktop/genome"
Error: Encountered internal HISAT2 exception (#1)
Command: /hisat2.1/HISAT2~1 -x C:/Users/Almunthir/Desktop/genome -U C:/Users/Almunthir/Desktop/SRR26264823_trimmed.fastq -S C:/Users/Almunthir/Desktop/SRR26264823_aligned.sam

could you plesaes help me with that and tell me is the formatting good this time ,I am new here so i am still learning things out

[BioinfGuru]

!!

[GenoMax]

No, I did not make the index myself. I downloaded the pre-built index files for GRCh38 from a reliable source.

Since you are using windows version of hisat2, it must be the 7 yr old v.2.1.0 (no newer builds appear to be available). So that may be your main issue. The index you downloaded was likely built for newer versions of hisat2 and is not compatible with the windows version. Thus it is not being recognized.

You could try and build the index yourself but you are going to be fighting a losing battle. On windows you are bound to run into some other issue with software assuming you have an adequate amount of RAM available to run this alignment. Using WSL2 (linux subsystem for windows) may be the way to go if you do not have access to alternate hardware and you are using Windows 11.

I am also not sure why you are trying to run this through R which adds another layer of complexity.

[altobialmunthir]

Thanks for the reply. I will attach the images of the preindexed GRch338 from HISAT2 and the Binaries that I've downloaded

so on the two above images I have downloaded windows version ,or it is still an issue ? if this persists to fail my only choice is WSL2 any other advices?

[GenoMax]

I have downloaded windows version ,or it is still an issue ?

The windows version of the program is incompatible with the index above (which must be for the latest hisat2).