**0**wrote:

Dear Sir / Madam,

Greetings!

I am a PhD student, in an academic institute of India. I have the following

queries:

1) We can use expected count for calculation of log fold change, and

log counts per million using edgeR package of R. In this package library

size is calculated, which represents sum of expected count of each contigs.

Kindly help me to understand calculation of normalization factors.

2) Multiplying the normalization factor with library size we get

effective library size, and after that this effective library size is used

for the calculation of normalized expected count. Kindly help me to

understand how normalized expected count is calculated?

3) Kindly also explain how the TMM_normalized FPKM is calculated?

4) Here is a example data, you are kindly

requested to calculate the normalization factor for effective library size,

normalized expected count, log fold change, log counts per million and TMM

normalized FPKM. I feel I can grasp easily from the calculation done in the

for this calculation.

Sorry have to post this example data here only because unable to attach excel sheet.

Example data:

Matrix of expected count | ||

Sample A | Sample B | |

c989_g1_i1 | 457 | 134 |

c1001_g1_i1 | 482 | 117 |

c997_g1_i1 | 3 | 16 |

Matrix information after analysis | |||

group | lib.size | norm.factors | eff.lib.size |

Sample A | 942 | 1.016076654 | 957.1442 |

Sample B | 267 | 0.984177716 | 262.7755 |

I want to understand how this norm.factors are calculated?

Results of edgeR from the matrix | ||||

logFC | logCPM | PValue | FDR | |

c997_g1_i1 | 4.193418634 | 14.56202158 | 0.000305695 | 0.000917085 |

c1001_g1_i1 | -0.177541924 | 18.86089555 | 0.799344988 | 0.888664958 |

c989_g1_i1 | 0.094905219 | 18.91100806 | 0.888664958 | 0.888664958 |

Kindly help me to understand how normalised expected count is calculated?

Information for calculation of TMM_normalized FPKM | |||

group | lib.size | norm.factors | eff.lib.size |

Sample A | 3505040 | 1.041236155 | 3649574 |

Sample B | 3399608 | 0.960396924 | 3264973 |

For the calculation of TMM normalised FPKM, how this normalisation factor is calculated?

After that we get TMM normalised FPKM, which is

TMM_normalized_FPKM | ||

Sample A | Sample B | |

c989_g1_i1 | 412316.07 | 440363.64 |

c1001_g1_i1 | 1171119.49 | 1035458.31 |

c997_g1_i1 | 5958.79 | 115757.58 |

To process some of my transcriptome data of doctoral study, you are kindly

requested to please clarify the aforementioned doubts. I shall be highly

grateful for this help.

I look forward your response.

With regards

Ashish Kumar Pathak