A method of validating a change in an RNA expression pipeline through identification and removal of outliers from a batch dataset produced by the RNA expression pipeline, the method comprising:
- at a computer system having one or more processors, and memory storing one or more programs for execution by the one or more processors:
- a) obtaining, in electronic form, a batch dataset comprising, for each respective biological test sample in a plurality of biological test samples,
- a corresponding expression profile prepared using a first methodology, the corresponding expression profile comprising a corresponding gene expression value for each respective gene in a first set of genes, wherein obtaining the batch dataset comprises:
- obtaining, in electronic form, a corresponding plurality of at least 10,000 sequence reads derived from the respective biological test sample by RNA sequencing;
- and
- determining, from the corresponding plurality of at least 10,000 sequence reads, the corresponding gene expression value for each respective gene in the first set of genes, and
- a corresponding set of characteristic values comprising a respective characteristic value for each respective characteristic in a first set of characteristics about the respective biological test sample;
- b) determining, for the batch dataset, a reference cohort by a procedure comprising:
- (i) selecting a subset of reference samples from a plurality of reference samples, wherein, each respective reference sample in the reference cohort is associated with a corresponding set of characteristic values comprising a corresponding characteristic value for each respective characteristic in a second set of characteristics about the respective reference sample, wherein the second set of characteristics comprises a third set of one or more characteristics that are also present in the first set of characteristics,
- (ii) determining that the percentage of respective biological test samples, in the plurality of biological test samples, having a respective value for a first characteristic in the third set of one or more characteristics, fails to be within a threshold percentage of the percentage of respective reference samples, in the subset of reference samples, having the same respective value for the first characteristic, wherein two respective biological test samples in the plurality of biological test samples have different respective values for the first characteristic and wherein the threshold percentage is five percent,
- (iii) determining a subset of biological test samples that cause the percentage of respective biological test samples, in the plurality of biological test samples, having a respective value for the respective characteristic to fail to be within the threshold percentage of the percentage of respective reference samples, in the subset of reference samples, having the same respective value for the first characteristic,
- (iv) removing the subset of biological test samples from the plurality of biological test samples, and
- (v) determining that the percentage of respective biological test samples, in the plurality of biological test samples, having a respective value for the respective characteristic, is within the threshold percentage of the percentage of respective reference samples, in the subset of reference samples, having the same respective value for the first characteristic;
- c) obtaining, in electronic form, for each respective reference sample in the reference cohort,
- a corresponding expression profile comprising a corresponding gene expression value for each respective gene in the first set of genes prepared using a second methodology that is different than the first methodology;
- d) determining (i) a first distribution for the batch dataset, with the subset of biological test samples removed from the plurality of biological test samples, using the corresponding expression profile for each respective biological test sample in the plurality of biological test samples and (ii) a second distribution for the reference cohort using the corresponding expression profile for each respective reference sample in the subset of reference samples;
- and
- e) validating the change in the RNA expression pipeline on the basis that a comparison of the first distribution and the second distribution satisfies a comparison criterion.