The classifier

Welcome to the BRCA1-like MLPA classifier website!

This online tool helps you to classify a tumor sample as BRCA1-like or Sporadic-like, when the P376-BRCA1ness probe mix of MRC-Holland is used.

The BRCA1-like signature is derived from BRCA1-mutated tumors. The BRCA1-like aCGH pattern may also be present in sporadic breast cancers, which frequently show evidence of impaired BRCA1 function due to other causes than mutation, for example, methylation. We and others have shown that sporadic basal-like breast cancers resemble BRCA1 mutated cancers in many different ways. This concept of "BRCAness" in sporadic cancers is under study in many centres as it may have implications for treatment selection.

Documentation

Introduction MLPA classifier as Word document
Analysis protocol for genemapper data
Example of a genemapper data file
Example of a normalized data file
Probe info
Training data

Manuscript

Lips EH , Laddach N, Savola SP, Vollebergh MA, Oonk AM, Imholz AL, Wessels LF, Wesseling J, Nederlof PM, Rodenhuis S. Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness. Breast Cancer Res. 2011;13:R107.

Tool

This website is developed by Abderrahim Ajouaou, Lennart Mulder, Esther Lips and Jelle ten Hoeve.

Contact

For more information, contact Esther Lips or Jelle ten Hoeve .



1. Genemapper data

Input should be the output of Genemapper (or any other fragment analysis software program) with the samples in rows and the height and peak areas as columns.

Explanation

This input file should contain between 3 and 10 Reference samples (reference samples should be named R1, R2, R3, etc.) and peak heights (for QC) and peak areas (assay probes). The peaks should be named Peak.Height 1…till Peak. Area 54. Be aware that the probes with length 255, 445 and 500 are of insufficient quality and should be removed in the genemapper output. The first 9 probes (or Peak Heights) are for quality control. Starting from probe 10 (Peak Area 10) are the assay probes. Check the example Raw input file carefully and be sure that your input file has exactly the same layout. The tool first checks the quality of the values by two quality checks (see also the general MLPA guidelines).

Quality Check 1

If the peak heights of the Q-fragments (64,70,76,82 nt) (Peak.Heights 1,2,3,and 4) are all higher than 1/3 of the 92nt (Peak.Height 6) fragment, than the quantity of the input DNA is not sufficient, or the ligation step failed. Repeat the experiment for this sample with sufficient DNA.

Quality Check 2

If one of the D-fragments (88, 96 nt) is much lower (<40%) than the 92nt fragment, the denaturation of the DNA failed. Repeat the experiment for this sample.

Quality Check 3

This check estimates the degree of signal sloping in the peak pattern of MLPA probes. The average signal intensity of the longest fragments (Peak.Height 10, 11, 12, 13, and 14) should be at least 1/3rd of that of the shorter fragments (Peak.Height 50, 51, 52, 53, and 54); this is an acceptable sloping.


2. Normalised data

This sheet contains the normalized MLPA data, either from step 1 (Genemapper data) or your own normalized data.

Explanation

This sheet contains the normalized MLPA data. In the first normalization step the probes are divided by the sum of all probes for that specific sample. In the second normalization step each probe value per sample is divided by the mean value of the reference probes. After normalization, all 34 BRCA1-like classification probes are put together into this “Pam input“ file. Alternatively, if you have performed the normalization yourself, you can upload your normalized data here. In this case, you should check that your PAM input file has exactly the same lay-out as the example Normalized Data.


3. Classification results

This sheet contains the BRCA1-like classification result.

Explanation

This sheet contains the BRCA1-like classification. Each sample gets a BRCA1-like and a Sporadic-like score, we call a sample with a BRCA1-like score of 0.5 or higher BRCA1-like. This sheet also contains the results of the quality control.