Quality control templates ensuring validity of sequencing-based assays

Abstract

Embodiments of a method and/or system can include generating a set of quality control template (QCT) molecules; determining a set of QCT sequence read clusters based on the set of QCT molecules, such as based on variation regions of the set of QCT molecules; and based on the set of QCT sequence read clusters, determining a sequencing-related parameter, such as a contamination parameter and/or molecule count parameter, associated with the at least one of sequencing library preparation and sequencing.

We claim: 
     
         1 . A method for facilitating prenatal diagnosis of a genetic disorder from a maternal sample associated with a pregnant woman, the method comprising:
 adding, to the maternal sample, a set of quality control template (QCT) molecules associated with the genetic disorder, the set of QCT molecules comprising:
 target-associated regions with sequence similarity to a target sequence region of endogenous target molecules, and 
 variation regions with sequence dissimilarity to a sequence region of the endogenous target molecules; 
   generating a co-amplified mixture based on co-amplifying the set of QCT molecules and nucleic acid molecules comprising the target sequence region;   sequencing the co-amplified mixture;   computationally determining a unique number of the set of QCT molecules, based on a number of the variation regions that are distinct and detected from QCT molecule sequence reads from the sequencing, wherein the QCT molecule sequence reads correspond to the set of QCT molecules;   calculating the average QCT sequencing depth based on dividing a number of the QCT molecule sequence reads by the unique number of QCT molecules;   determining an absolute count of the endogenous target molecules based on dividing a total read count for the endogenous target molecules by the average QCT sequencing depth;   determining an absolute count of endogenous reference molecules based on dividing a total read count for the endogenous reference molecules by the average QCT sequencing depth; and   facilitating the prenatal diagnosis of the genetic disorder based on a comparison between the absolute count of endogenous target sequences and the absolute count of endogenous reference sequences.   
     
     
         2 . The method of  claim 1 ,
 wherein the genetic disorder comprises a single gene disorder,   wherein determining the absolute count of the endogenous target molecules comprises determining the absolute count of the endogenous target molecules comprising a mutation associated with the single gene disorder, based on dividing the total read count for the endogenous target molecules by the average QCT sequencing depth,   wherein determining the absolute count of the endogenous reference molecules comprises determining the absolute count of the endogenous reference molecules lacking the mutation, based on dividing the total read count for the endogenous reference molecules by the average QCT sequencing depth, and   wherein facilitating the prenatal diagnosis of the genetic disorder comprises facilitating the prenatal diagnosis of the single gene disorder based on the comparison.   
     
     
         3 . The method of  claim 1 ,
 wherein the genetic disorder comprises a chromosomal abnormality,   wherein determining the absolute count of the endogenous target molecules comprises determining the absolute count of the endogenous target molecules associated with a first chromosome, based on dividing the total read count for the endogenous target molecules by the average QCT sequencing depth,   wherein determining the absolute count of the endogenous reference molecules comprises determining the absolute count of the endogenous reference molecules associated with a second chromosome, based on dividing the total read count for the endogenous reference molecules by the average QCT sequencing depth, and   wherein facilitating the prenatal diagnosis of the genetic disorder comprises facilitating the prenatal diagnosis of the chromosomal abnormality based on the comparison.   
     
     
         4 . The method of  claim 1 , wherein the genetic disorder comprises a chromosomal microdeletion,
 wherein determining the absolute count of the endogenous target molecules comprises determining the absolute count of the endogenous target molecules associated with a microdeletion region, based on dividing the total read count for the endogenous target molecules by the average QCT sequencing depth,   wherein determining the absolute count of the endogenous reference molecules comprises determining the absolute count of the endogenous reference molecules associated with a second chromosomal region not expected to have a microdeletion, based on dividing the total read count for the endogenous reference molecules by the average QCT sequencing depth, and   wherein facilitating the prenatal diagnosis of the genetic disorder comprises facilitating the prenatal diagnosis of the chromosomal microdeletion based on the comparison.   
     
     
         5 . The method of  claim 1 , wherein the genetic disorder comprises a copy number variation,
 wherein determining the absolute count of the endogenous target molecules comprises determining the absolute count of the endogenous target molecules associated with a region that may have copy number variation, based on dividing the total read count for the endogenous target molecules by the average QCT sequencing depth,   wherein determining the absolute count of the endogenous reference molecules comprises determining the absolute count of the endogenous reference molecules associated with a region not expected to have a copy number variation, based on dividing the total read count for the endogenous reference molecules by the average QCT sequencing depth, and   wherein facilitating the prenatal diagnosis of the genetic disorder comprises facilitating the prenatal diagnosis of the copy number variation based on the comparison.   
     
     
         6 . The method of  claim 1 , wherein the average QCT sequencing depth used in determining the absolute count of the endogenous target molecules and the absolute count of endogenous reference molecules is determined separately from their corresponding QCTs. 
     
     
         7 . The method of  claim 1 , wherein facilitating the prenatal diagnosis comprises facilitating the prenatal diagnosis of the genetic disorder based on a fetal fraction measurement, the absolute count of endogenous target sequences, and the absolute count of endogenous reference sequences. 
     
     
         8 . A method for identifying contamination associated with at least one of sequencing library preparation and high throughput sequencing, the method comprising:
 generating a set of quality control template (QCT) molecules, each QCT molecule comprising:
 a target-associated region with sequence similarity to a target sequence region of a biological target, and 
 a variation region with sequence dissimilarity to a sequence region of the biological target; and 
   computationally determining a set of QCT sequence read clusters based on the variation regions of the set of QCT molecules,   wherein the set of QCT sequence read clusters comprises QCT molecule sequence reads derived from the high throughput sequencing corresponding to a set of QCT mixtures generated based on the set of QCT molecules and a set of samples comprising the biological target, and   wherein the sequencing library preparation comprises co-amplification, of the set of QCT molecules and nucleic acid molecules comprising the biological target, based on the sequence similarity of the target-associated region and the target sequence region of the biological target; and   based on the set of QCT sequence read clusters, determining a contamination parameter describing the contamination associated with the at least one of the sequencing library preparation and the high throughput sequencing.   
     
     
         9 . The method of  claim 8 , wherein computationally determining the set of QCT sequence read clusters comprises:
 clustering a first QCT molecule sequence read and a second QCT molecule sequence read into a QCT sequence read cluster, of the set of QCT sequence read clusters, based on a variation region sequence similarity satisfying a first condition, and   for each QCT sequence read cluster of the set of QCT sequence read clusters, determining an assignment of the QCT sequence read cluster to a sample identifier of a set of sample identifiers identifying the set of samples,   wherein determining the contamination parameter comprises determining the contamination parameter based on the set of QCT sequence read clusters and the assignments of the QCT sequence read clusters to the sample identifiers of the set of sample identifiers.   
     
     
         10 . The method of  Claim 9 , wherein determining the contamination parameter comprises:
 identifying a first and a second QCT sequence read cluster corresponding to a shared variation region sequence, wherein the assignments of the first and the second QCT sequence read clusters are to distinct sample identifiers of the set of sample identifiers;   generating a read depth comparison between a first read depth associated with the first QCT sequence read cluster and a second read depth associated with the second QCT sequence read cluster; and   based on the read depth comparison, determining the contamination parameter associated with a sample identified by a distinct sample identifier of the distinct sample identifiers.   
     
     
         11 . The method of  claim 9 , wherein clustering the first and the second QCT sequence reads comprises clustering the first and the second QCT sequence reads into the QCT sequence read cluster based on the variation region sequence similarity of fewer than three point substitutions, and based on a read depth associated with the QCT sequence read cluster satisfying a second condition. 
     
     
         12 . The method of  claim 8 , wherein determining the contamination parameter comprises:
 determining a first molecular fingerprint associated with first amplification in a first instance of the sequencing library preparation, based on the set of QCT sequence read clusters;   determining a second molecular fingerprint associated with second amplification in a second instance of the sequencing library preparation, based on an additional set of QCT sequence read clusters; and   based on a comparison between the first and the second molecular fingerprints, determining a carry-over contamination parameter describing carry-over contamination from the first instance to the second instance.   
     
     
         13 . The method of  claim 8 , wherein the contamination parameter describes a degree of index misassignment associated with the high throughput sequencing. 
     
     
         14 . The method of  claim 13 , wherein the contamination parameter is adapted for use in determination of a diagnostic outcome for assays associated with at least one of noninvasive prenatal testing and liquid biopsies. 
     
     
         15 . The method of  claim 8 , further comprising generating a single QCT library comprising the set of QCT molecules, wherein the single QCT library is adapted for deployment, at a single stage of the at least one of the sequencing library preparation and the high throughput sequencing, of less than 0.00001 nanograms of amplifiable QCT molecules for each sample of the set of samples. 
     
     
         16 . The method of  claim 8 ,
 wherein each variation region of the set of QCT molecules comprises an embedded molecular identifier (EMI) region comprising a set of variable “N” bases, wherein each “N” base is selected from any one of an “A” base, a “G” base, a “T” base, and a “C” base,   wherein each QCT molecule of the set of QCT molecules further comprises an additional EMI region comprising an additional set of variable “N” bases, wherein the additional EMI region is separated from the EMI region by a sequence region of the QCT molecule, wherein the set of variable “N” bases and the additional set of variable “N” bases each comprise greater than three “N” bases, and   wherein determining the contamination parameter comprises determining the contamination parameter based on the set of QCT sequence read clusters derived based on the EMI regions and the additional EMI regions of the set of QCT molecules.