How to handle large RNA-seq sets in STAR?

Pre-Processing the RNA-seq Data

• Start by ensuring your RNA-seq data is clean, which includes removing low-quality reads and adapters using tools like Trimmomatic or FastQC.

 

• Check the quality of the cleaned reads once again to ensure the data integrity and quality are up to standards before alignment.

Prepare Reference Genome

• Download the reference genome in FASTA format from a reliable source, such as Ensembl or NCBI.

 

• Ensure you also have the corresponding annotation file in GTF or GFF format, as this is crucial for accurate mapping and downstream analyses.

Create Genome Index

• Use STAR to generate an index for the reference genome. This is a prerequisite step before aligning your RNA-seq reads.

 

• The command usually looks like this:

  ```
  STAR --runThreadN --runMode genomeGenerate --genomeDir --genomeFastaFiles --sjdbGTFfile <path_to_annotation_gtf>
  ```

  Adjust the paths and number of threads according to your setup.

Align RNA-seq Reads

• Run STAR to align your RNA-seq reads to the reference genome using the index created earlier. Use the following command as a framework:

  ```
  STAR --runThreadN --genomeDir --readFilesIn --outFileNamePrefix
  ```

  Adjust the relevant parameters if you are processing paired-end reads. Specify the number of threads to make use of available computational resources efficiently.

 

• If the reads are compressed, remember to include the `--readFilesCommand zcat` option or the appropriate command to decompress your files on the fly.

Handling Large Datasets

• Divide the input files if they are too large. This can often help in managing memory usage and computational load.

 

• Opt for a machine with ample RAM and use multiple cores to speed up the process through parallelization, especially for large datasets.

Post-Processing Analysis

• After the alignment, examine the output files. The important files typically include `Aligned.out.sam`, which contains the alignment results.

 

• Convert the SAM file to a BAM file using `samtools`, which reduces the file size and speeds up downstream processing:

  ```
  samtools view -bS Aligned.out.sam > Aligned.out.bam
  ```

  Sort and index the BAM file to efficiently query your alignments:

 

• Use the following commands to sort and index:

  ```
  samtools sort Aligned.out.bam > Aligned.sorted.bam
  samtools index Aligned.sorted.bam
  ```

Evaluate the Alignment

• Use tools like `Qualimap` or `RNA-SeQC` to assess the quality of your RNA-seq alignment. This step is crucial to ensure the fidelity and reliability of your data for subsequent analysis.