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 comparison of gene expression levels across different samples than FPKM alone.

2. How Does the Calculator Work?

The calculator uses the TPM formula:

Where:

• \( FPKM \) — Fragments Per Kilobase Million value
• \( \text{Gene Length} \) — Gene length in base pairs
• \( \sum(FPKM \times \text{Gene Length} / 1000) \) — Sum of all (FPKM × Gene Length / 1000) values in the sample

Explanation: The formula normalizes FPKM values by accounting for the total transcriptome output, making expression levels comparable across different samples.

3. Importance of TPM Normalization

Details: TPM normalization is crucial for accurate comparison of gene expression levels between different samples and experiments. It corrects for variations in gene length and sequencing depth, providing more reliable quantitative results.

4. Using the Calculator

Tips: Enter FPKM value, gene length in base pairs, and the sum of all (FPKM × Gene Length / 1000) values from your sample. All values must be positive numbers.

5. Frequently Asked Questions (FAQ)

Q1: What's the difference between TPM and FPKM?
A: While both normalize for gene length and sequencing depth, TPM additionally normalizes for the total transcriptome output, making it more suitable for sample comparisons.

Q2: When should I use TPM instead of FPKM?
A: Use TPM when you need to compare expression levels across different samples or experiments, as it provides better cross-sample comparability.

Q3: How do I calculate the sum of (FPKM × Gene Length / 1000)?
A: This requires calculating (FPKM × Gene Length / 1000) for all genes in your sample and summing these values together.

Q4: Are there limitations to TPM normalization?
A: TPM works well for most applications but may not be ideal for detecting differential expression in very lowly expressed genes or when comparing across different species.

Q5: Can I convert TPM back to FPKM?
A: Yes, but you would need the original sum value used in the TPM calculation to reverse the normalization.