Molecular quality assurance methods for use in sequencing

Abstract

The present invention relates to quality assurance methods for use in amplification techniques, such as Next Generation Sequencing (NGS).

1 . A method of determining the complexity of a nucleic acid template, or of identifying a true sequence variant, comprising:
 i) providing a nucleic acid template;   ii) providing a plurality of primer pairs, comprising a first primer and a second primer, wherein the first primer comprises a sequence complementary to a portion of the nucleic acid template, and the second primer comprises a sequence complementary to a portion of the complement of the nucleic acid template;   iii) attaching a codeword to the 5′ end of the first primer, the 5′ end of the second primer, or both, to form a codeword-primer molecule, or to the nucleic acid template to form a codeword-template molecule;   iv) performing an amplification reaction with the paired codeword-primer molecules and the nucleic acid template or with the primer pairs and the codeword-template molecule for a defined number of cycles to obtain an amplification reaction product;   v) obtaining the sequence of the amplification reaction product at the end of each cycle, at the end of the defined number of cycles, or at an intermediate number of cycles;   vi) determining the abundance of each codeword present in the amplification reaction product at the end of each cycle, at the end of the defined number of cycles, or at an intermediate number of cycles;   vii) determining the observed codeword entropy of each cycle; and   viii) comparing the observed codeword entropy to an estimated codeword entropy, to determine the complexity of the nucleic acid template; or   ix) performing a supervised classification method based on the results of steps vi) and vii), to identify the true sequence variant.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1  wherein the true sequence variant is a low prevalence sequence variant. 
     
     
         4 . The method of  claim 1  wherein the nucleic acid template is a DNA template. 
     
     
         5 . The method of  claim 1  wherein the codeword-primer molecule or the primer is further attached to an adapter sequence. 
     
     
         6 . The method of  claim 1  wherein a different codeword is attached to each primer in the primer pair or the same codeword is attached to each primer in the primer pair. 
     
     
         7 . The method of  claim 1  wherein the codewords are attached to the nucleic acid template at random. 
     
     
         8 . The method of  claim 1  wherein the observed codeword entropy is calculated by Shannon entropy, the Simpson index, or any other diversity index. 
     
     
         9 . The method of  claim 1  wherein the codewords are present in a non-uniform pool or are present in a balanced pool. 
     
     
         10 . (canceled) 
     
     
         11 . A method for obtaining a balanced pool of codewords comprising:
 i) providing an initial sample comprising a plurality of codewords of a defined length;   ii) providing a target sequence;   iii) providing a plurality of primer pairs comprising a first primer and a second primer, wherein the first primer comprises a sequence complementary to a portion of the target sequence, and the second primer comprises a sequence complementary to a portion of the complement of the target sequence, and wherein each codeword is attached to the 5′ end of the first primer, the 5′ end of the second primer, or both, to form a paired codeword-primer molecule;   iv) performing an amplification reaction with the paired codeword-primer molecule and the target sequence for a defined number of cycles to obtain an amplification reaction product;   v) obtaining the sequence of the amplification reaction product at the end of each cycle, at the end of the defined number of cycles, or at an intermediate number of cycles;   vi) determining the abundance of each codeword present in the amplification reaction product at the end of each cycle, at the end of the defined number of cycles, or at an intermediate number of cycles;   vii) obtaining measured parameters of codeword performance by:
 a) comparing the abundance from step (vi) with an expected number; and/or 
 b) determining the rate of increase in abundance over each preceding amplification cycle; and 
   
       using the measured parameters from step (vii) to perform a search in silico using a stochastic local search method to obtain a balanced pool of codewords. 
     
     
         12 . The method of  claim 11  wherein the codeword-primer molecule is further attached to an adapter molecule. 
     
     
         13 . The method of  claim 11  wherein the defined length is from about 4 units to about 21 units. 
     
     
         14 . The method of  claim 11  wherein the initial sample: comprises at least 10 codewords; is a random sample; is subjected to combinatorial constraints; is subjected to thermodynamic constraints; comprises all combinations of codeword sequences; comprises a subset of combinations of codeword sequences; or a combination thereof. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 11  wherein the method is performed using larger pools of codewords or codewords of different lengths or using a single target sequence or using two or more target sequences. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 11  wherein the method is performed a single time or is performed two or more times. 
     
     
         20 . The method of  claim 11  comprising determination of codeword performance as function of subsequence and location. 
     
     
         21 . The method of  claim 11  further comprising determining one or more of amplification process, contamination, sample identity mismatch, or codeword pool imbalance. 
     
     
         22 . A primer comprising one or more of the sequences set forth in SEQ ID NOs: 1-146. 
     
     
         23 . The primer of  claim 22 , wherein the primer is provided as a set of primer pairs, comprising a first primer and a second primer, wherein the first primer comprises a sequence set forth in any one of SEQ ID NOs: 1-73 and the second primer comprises a sequence set forth in any one of SEQ ID NOs: 74-146. 
     
     
         24 .) The primer of  claim 22 , wherein the primer is provided in a kit that further comprises suitable reagents for storage, transport, delivery or use of the primers or primer pairs, optionally with instructions for use. 
     
     
         25 . The method of  claim 1  further comprising determining one or more of amplification process, contamination, sample identity mismatch, or codeword pool imbalance.