How to troubleshoot low HISAT2 alignment rates?

Check Input Files Quality

• Ensure your FASTQ files are of high quality. Use tools like FastQC to assess quality issues such as poor base quality or adapters.

 

• If quality is an issue, consider trimming reads using software like Trimmomatic or Cutadapt to remove low-quality bases or adapters.

Verify Index and Reference Files

• Check if the HISAT2 index was built correctly. Ensure you are using the right reference genome version matching your experiment.

 

• Rebuild the index with HISAT2 if any doubts arise on its integrity. Use the correct genome fasta file.

Adapter and Quality Trimming

• Perform adapter trimming if adapters are still present. This can greatly improve alignment rates.

 

• Reassess read quality post-trimming to ensure that important data is not being discarded.

Check Paired-End Read Order

• Ensure paired-end reads are properly ordered and synced. Mismatched read pairs can affect mapping rates.

 

• Tools like repair.sh from the BBTools suite can be useful to fix order mismatches.

Review Alignment Parameters

• Ensure that you're using appropriate HISAT2 parameters for your read length and quality. Parameters such as `--max-intronlen` might need adjusting.

 

• Experiment with `--end-to-end` or `--local` modes to see if one yields better alignment rates for your data characteristics.

Consult Log Files

• Review HISAT2 log files to understand unaligned reads. They provide clues like too many mismatches or lacking alignment scores.

 

• Assess any reported errors or warnings and address them based on the recommendations provided by HISAT2.

Use Alternative Splicing Awareness

• HISAT2 is designed for splicing-aware alignments; ensure that your input data has the expected splicing patterns.

 

• Consider using known splice site files with the `--known-splicesite-infile` option if applicable to your study.

Validate Output File Compatibility

• Ensure that the output alignment file is compatible with downstream analysis tools, and no conversion issues occur.

 

• Check the output format and possibly convert it to the necessary format using tools like SAMtools.

Seek Community Help

• If alignment issues persist, consider reaching out to user forums or communities with details of the problem and steps already taken.

 

• Posting logs and command lines can help others offer more specific troubleshooting advice.