Methods and compositions for preparing polynucleotide libraries

Abstract

Provided herein are methods of producing molar amounts of nucleic acids in a sample by using fixed molar amounts of primers and amplifying nucleic acids in the sample through a sufficient number of rounds to use all the primer. The methods may be used on a plurality of separate samples to produce samples with molar amounts of nucleic acids that are the same, or substantially the same, or that are related in known molar ratios. Further provided herein are normalized, optionally pooled nucleic acid libraries.

What is claimed is: 
     
         1 . A method of amplifying nucleic acids comprising
 a) performing a plurality of primer extension reactions on nucleic acid molecules in each of a plurality of separate nucleic acid samples containing nucleic acids, using first polynucleotide primers to initiate primer extension in each sample, wherein:
 1) each of the plurality of samples is provided with a fixed molar amount of first primer; 
 2) the fixed molar amount of first primer in each sample is the same or substantially the same, or is related to the molar amount in other samples in a known ratio; and 
 3) the plurality of primer extension reactions in each sample is such that, upon completion of the plurality of primer extension reactions, all first primer in each sample has been used to produce amplified nucleic acids. 
   
     
     
         2 . The method of  claim 1  further comprising using a second polynucleotide primer, wherein the second primer is present in equal or substantially equal molar amount as the first primer, or in a known greater molar amount as the first primer. 
     
     
         3 . The method of  claim 2  wherein the second primer is present in equal or substantially equal molar amount as the first primer, and after the plurality of primer extension reactions the molar amount of amplified nucleic acid is equal to or substantially equal to the molar amount of first and/or second primer. 
     
     
         4 . The method of  claim 2  wherein the first and second primers are different. 
     
     
         5 . The method of  claim 1  further comprising
 b) combining a portion of each of the plurality of separate samples to provide a pooled sample comprising a library of amplified nucleic acids, wherein the molar amount of amplified nucleic acid from each separate sample in the pooled sample is the same or substantially the same, or related to the molar amounts of other nucleic acids in the pooled sample in a known ratio, 
 
       thus producing a pooled nucleic acid library. 
     
     
         6 . The method of  claim 1  wherein the molar amount of first primer in each sample is the same or substantially the same. 
     
     
         7 . The method of  claim 1  wherein the nucleic acid molecules in the plurality of separate samples comprise RNA molecules. 
     
     
         8 . The method of  claim 1  wherein the amplified nucleic acids comprise a sample barcode comprising a predetermined nucleotide sequence, wherein the barcode is different for each different nucleic acid sample. 
     
     
         9 . A method for amplifying nucleic acids comprising:
 a) providing a plurality of samples comprising RNA;   b) performing a first round of cDNA synthesis on the RNA in the samples to produce a library of first strand cDNA molecules;   c) performing a second round of cDNA synthesis on the cDNA strand produced in the first round to provide second strand cDNA molecules, wherein the second strand cDNA molecules comprise a first primer binding site for a first primer and a template for a second primer binding site for a second primer; and   d) amplifying the cDNA produced in step c) by providing each of the plurality of samples a molar amount of first primer and second primer, wherein the molar amount of the second primer is equal to or substantially equal to the molar amount of the first primer, or is greater than the molar amount of the first primer in a known amount, and performing a plurality of primer extension reactions, wherein
 1) the molar amount of first primer in each of the plurality of samples is the same or substantially the same, or is related to the molar amount in other samples in a known ratio; and 
 2) wherein the molar amount of first primer in each sample is such that, upon completion of the plurality of primer extension reactions, all first primer in each sample has been used. 
   
     
     
         10 . The method of  claim 9  wherein the first primer comprises a first polynucleotide sequence and the second primer comprises a second polynucleotide sequence, wherein the first and second polynucleotide sequences are the same, and wherein the molar amount of first and second primer in each sample is the same. 
     
     
         11 . The method of  claim 9  wherein at least one of the primers comprises sequencing platform-specific adapter sequences. 
     
     
         12 . The method of  claim 11  wherein the sequencing platform specific sequences comprise one or more of a sequencing primer hybridization site, a sample barcode and a cluster primer binding site. 
     
     
         13 . The method of  claim 9  further comprising:
 e) preparing a pooled nucleic acid library by combining a portion of each of the plurality of samples comprising amplified nucleic acid. 
 
     
     
         14 . The method of  claim 9  wherein the predetermined molar amount of first primer in each of the plurality of samples is the same or substantially the same. 
     
     
         15 . A method comprising
 a) preparing a set of nucleic acid libraries by amplifying nucleic acids in a plurality of separate samples comprising nucleic acids so that the amplified nucleic acids are present in each of the plurality of separate samples in the same or substantially the same molar amount, or are present in molar amounts in known ratios to each other; and   b) combining a portion of each of the separate samples to produce a pool of nucleic acid libraries in which the molar amounts of amplified nucleic acids from each nucleic acid library in the pool are the same or substantially the same, or are present in molar amounts in known ratios to each other.   
     
     
         16 . The method of  claim 15  further comprising:
 c) sequencing the nucleic acids in the pool of nucleic acid libraries. 
 
     
     
         17 . The method of  claim 15  wherein the amplified nucleic acids in the separate samples comprises a barcode, wherein the barcode for any nucleic acid library is the same for nucleic acids in that library and different from the barcodes for other nucleic acid libraries. 
     
     
         18 . A set of a plurality of separate nucleic acid libraries, wherein:
 a) each library comprises polynucleotides; and   b) molar amounts of polynucleotide molecules from each nucleic acid library in the set are the same or substantially the same, or are related as a known ratio to each other.   
     
     
         19 . The set of nucleic acid libraries of  claim 18 , wherein each separate nucleic acid library is in a separate container. 
     
     
         20 . The set of nucleic acid libraries of  claim 18  further wherein
 b) the polynucleotides comprise a sample barcode, and polynucleotides in any library have the same sample barcode; and 
 c) sample barcodes in different libraries are different. 
 
     
     
         21 . The set of nucleic acid libraries of  claim 18  wherein the molar amounts of polynucleotide molecules in each library are the same or substantially the same.