1. What is TPM Calculation?

TPM (Transcripts Per Million) is a normalization method for RNA-seq data that accounts for both gene length and sequencing depth. It provides a more accurate representation of gene expression levels compared to RPKM.

2. How Does the Calculator Work?

The calculator uses the TPM formula:

Where:

• \( RPKM \) — Reads Per Kilobase Million value for a specific gene
• \( \sum RPKM \) — Sum of all RPKM values in the sample
• \( 10^6 \) — Scaling factor to get per million transcripts

Explanation: This conversion ensures that the sum of all TPM values in a sample equals 1 million, making different samples directly comparable.

3. Importance of TPM Calculation

Details: TPM normalization is crucial for accurate gene expression analysis, enabling fair comparisons between samples and eliminating biases from different sequencing depths and gene lengths.

4. Using the Calculator

Tips: Enter the RPKM value for your gene of interest and the sum of all RPKM values in your sample. Both values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: Why convert RPKM to TPM?
A: TPM provides better between-sample comparability as it normalizes for the total transcript count in each sample.

Q2: What's the difference between RPKM and TPM?
A: While both account for gene length and sequencing depth, TPM normalizes by the total TPM sum per sample (1 million), making sample comparisons more straightforward.

Q3: When should I use TPM instead of RPKM?
A: TPM is generally preferred for comparing expression levels across different samples, while RPKM is more suitable for within-sample comparisons.

Q4: Are there limitations to TPM calculation?
A: TPM assumes uniform transcript distribution and may not account for all technical variations in RNA-seq data.

Q5: How do I calculate the sum of RPKM values?
A: The sum of RPKM values is calculated by adding up all individual RPKM values for all genes/transcripts in your RNA-seq sample.