Introduction:

This tool takes a peak table file as input and performs normalization based on quality control samples (QCs).

QCs are pooled samples. They contain the same compounds as the subject samples and are supposed to reflect the average metabolite concentrations within a study. QCs are pretreated according to the same protocols as the subject samples and are evenly injected throughout the analyses. The performances of the pretreatment and the analytical platform can be assessed using the QCs. The normalization formula is (x metabolite/QC metabolite) *10000.

Input files:

1. Peak table file in Tab-delimited txt format, with the first column as the compound identifier and others as samples.

For example:

AlignID	STDmix_GC_01	STDmix_GC_02	QC1	STDmix_GC_03	QC2
1	1486892478	561322777	3448620272	3448620272	561322777
Nitrogen dioxide	5492977592	684434115	3265669981	3265669981	3265669981
Ethanol, 2-fluoro-	2265686433	4182838129	4365291513	4365291513	4182838129
3-Pentanone, 2,2,4,4-tetramethyl-	13390154	12612932	21155307	21155307	21155322
Hydrazine	14588107	8510918	7224351	7224351	7224380

2. Sample-to-QC design file, Tab-delimited text file with two columns, "sample" and "QC".

For example:

STDmix_GC_01	QC1
STDmix_GC_02	QC1
STDmix_GC_03	QC2

Output files:

1. 'QC_norm_pkTable.txt', normalized peak table.

For example:

AlignID	STDmix_GC_01	STDmix_GC_02	QC1	STDmix_GC_03	QC2
1	4311.558	1627.673	10000	61437.38	10000
Nitrogen dioxide	16820.37	2095.846	10000	10000	10000
Ethanol, 2-fluoro-	5190.229	9582.036	10000	10436.2	10000
3-Pentanone, 2,2,4,4-tetramethyl-	6329.454	5962.065	10000	9999.993	10000
Hydrazine	20192.97	11780.88	10000	9999.96	10000

Note:

Experimental preparation: QCs must be pretreated according to the same protocols as the subject samples and are evenly injected throughout the analyses. Systematic nonbiological error is reduced by evenly placing them among subject samples.

For example:

sample1, sample2, sample3, QC1, saple4, sample5, QC2…QC m-1, sample n-2, sample n-1, sample n, QC m

Here a right “Sample-to-QC design file” must be prepared by users. The form is two columns, "sample " and "QC ", like before example given. STDmix_GC_01, STDmix_GC_02 correspond to QC1; STDmix_GC_03 corresponds to QC2.

For example:

STDmix_GC_01	QC1
STDmix_GC_02	QC1
STDmix_GC_03	QC2

Reference:

[1] Chen T, Tian G, Quan W. Pooled sample-based workflow and software for medical data calibration[C]// International Symposium on Medical Information and Communication Technology. IEEE, 2014:1-4.