Analytical standards and methods of using same

Abstract

Analytical standards can allow one to detect and/or measure sampling, processing, and/or amplification errors in a sample that includes a plurality of polynucleotide molecules. The analytical standards can provide an internal control to detect errors in the representation of the original sample reflected in data obtained after manipulating and/or processing of sample molecules.

1 - 20 . (canceled) 
     
     
         21 . A method of measuring abundance values among a plurality of polynucleotides, the method comprising:
 obtaining a plurality of polynucleotides in a sample, the sample comprising:
 a first synthetic polynucleotide comprising a first PCR-free quantitation tag, the first PCR-free quantitation tag positioned between cut sites of a restriction enzyme; and 
 a second synthetic polynucleotide comprising a second PCR-free quantitation tag, the second PCR-free quantitation tag positioned between cut sites of a restriction enzyme; 
   digesting the first synthetic polynucleotide to liberate the first PCR-free quantitation tag;   digesting the second synthetic polynucleotide to liberate the second PCR-free quantitation tag;   sequencing the first PCR-free quantitation tag and the second PCR-free quantitation tag; and   measuring the abundance of the first PCR-free quantitation tag and the second PCR-free quantitation tag.   
     
     
         22 . The method of  claim 21  further comprising comparing the abundance of the first PCR-free quantitation tag and the abundance of the second PCR-free quantitation tag; and
 determining the relative abundance of the first synthetic polynucleotide and the second synthetic polynucleotide. 
 
     
     
         23 . The method of  claim 21 , wherein the first PCR-free quantitation tag is positioned between cut sites of the same restriction enzyme as the restriction enzyme whose cut sites the second PCR-free quantitation tag is positioned. 
     
     
         24 . The method of  claim 21 , wherein:
 the first PCR-free quantitation tag is positioned between cut sites of a first restriction enzyme; and   the second PCR-free quantitation tag is positioned between cut sites of a second restriction enzyme that differs from the first restriction enzyme.   
     
     
         25 . A method for detecting sub-sampling error in a sample comprising a plurality of polynucleotides, the method comprising:
 obtaining a sample comprising at least a first sample polynucleotide and a second sample polynucleotide;   spiking the sample with at least one synthetic diversity standard designed to detect sub-sampling error;   amplifying polynucleotides in the spiked sample;   sequencing a first sample polynucleotide, a second sample polynucleotide, and at least one synthetic diversity standard;   measuring the diversity of unique sequences of the synthetic diversity standard polynucleotide;   comparing the diversity of unique sequences of the synthetic diversity standard polynucleotide to an expected diversity of unique sequences of the synthetic diversity standard polynucleotide; and   detecting and quantifying sub-sampling error in the sample if the measured diversity of the synthetic diversity standard is less than the expected diversity of the synthetic diversity standard polynucleotide.   
     
     
         26 . The method of  claim 25  wherein the at least one synthetic diversity standard comprises a set of synthetic diversity standards. 
     
     
         27 . The method of  claim 22 , wherein the first PCR-free quantitation tag is positioned between cut sites of the same restriction enzyme as the restriction enzyme whose cut sites the second PCR-free quantitation tag is positioned. 
     
     
         28 . The method of  claim 22  wherein:
 the first PCR-free quantitation tag is positioned between cut sites of a first restriction enzyme; and 
 the second PCR-free quantitation tag is positioned between cut sites of a second restriction enzyme that differs from the first restriction enzyme.