Sequencing Adapter Manufacture and Use

Abstract

Technology provided herein relates in part to methods, processes, machines and apparatuses for determining sequences of nucleotides for nucleic acid templates in a nucleic acid sample. The technology provide herein also relates in part to methods, processes, machines and apparatuses for counting nucleic acid templates. Nucleic acid templates of a sample are tagged with nonrandom oligonucleotide adapters that include predetermined non-randomly generated sequences. The use of these nonrandom oligonucleotide adapters provides an efficient method to reduce sequencing errors, and increase the sensitivity of detection of low-frequency single nucleotide alterations.

1 . A method for determining a sequence of nucleotides for one or more nucleic acid templates in a nucleic acid sample, comprising:
 contacting double-stranded nucleic acid templates of the nucleic acid sample with partially double-stranded nonrandom oligonucleotide adapter species under ligation conditions, thereby generating adapter-ligated nucleic acid templates, wherein:
 each of the nonrandom oligonucleotide adapter species comprises a first oligonucleotide species and a second oligonucleotide species; 
 each of the first oligonucleotide species comprises 5′ to 3′ a polynucleotide A and a 5′-3′ polynucleotide B species and each of the second oligonucleotide species comprises 5′ to 3′ a polynucleotide B′ species and a 5′ to 3′ polynucleotide A′; 
 each of the polynucleotide B species and the polynucleotide B′ species are predetermined, are non-randomly generated, are the same length, and are about 4 to about 20 consecutive nucleotides in length; 
 there are 300 or fewer polynucleotide B species and each polynucleotide B′ species is a reverse complement of a polynucleotide B species; 
 polynucleotide A is not a reverse complement of polynucleotide A′; 
 the ratio of nucleic acid templates to polynucleotide B species is greater than 1,000 to 1; 
 the polynucleotide B species anneal to the complementary polynucleotide B′ species and the polynucleotide A′ species does not anneal to the polynucleotide A species; 
   amplifying the adapter-ligated nucleic acid templates, thereby generating amplicons; and   sequencing all or a portion of each amplicon, thereby determining a sequence of nucleotides for the one or more nucleic acid templates in the nucleic acid sample.   
     
     
         2 - 45 . (canceled) 
     
     
         46 . A composition comprising a plurality of partially double-stranded nonrandom oligonucleotide adapter molecules, wherein each of the nonrandom oligonucleotide adapter species comprises:
 a first oligonucleotide species and a second oligonucleotide species, wherein each of the first oligonucleotide species comprises 5′ to 3′ a polynucleotide A and a 5′ to 3′ polynucleotide B species;   each of the second oligonucleotide species comprises 5′ to 3′ a polynucleotide B′ species and a 5′ to 3′ polynucleotide A′ species; and   wherein each of the polynucleotide B species and the polynucleotide B′ species are the reverse complement of each other and each of the polynucleotide A species is not a reverse complement of polynucleotide A′ species,   each of the nonrandom oligonucleotide adapter species comprises a first oligonucleotide species and a second oligonucleotide species;   each of the first oligonucleotide species comprises a polynucleotide A species and a polynucleotide B species and each of the second oligonucleotide species comprises a polynucleotide B′ species and a polynucleotide A′ species;   each of the polynucleotide B species and the polynucleotide B′ species are predetermined, are non-randomly generated and are about 4 to about 20 consecutive nucleotides in length;   there are 999 or fewer polynucleotide B species and each polynucleotide B′ species is a reverse complement of a polynucleotide B species;   each polynucleotide A species is not a reverse complement of polynucleotide A′ species;   the ratio of the double-stranded nucleic acid template species to the polynucleotide B species is greater than 1,000 to 1;   the polynucleotide B species anneal to the complementary polynucleotide B′ species and the polynucleotide A′ species does not anneal to the polynucleotide A species.   
     
     
         47 . The composition of  claim 46 , wherein the polynucleotide B species and the polynucleotide B′ species are predetermined sequences and are non-randomly generated. 
     
     
         48 . The composition of  claim 46 , wherein the individual pairs of polynucleotide B species and the polynucleotide B′ species are the same length. 
     
     
         49 . The composition of  claim 46  wherein the individual pairs of the polynucleotide B species and the polynucleotide B′ species are the same length but different polynucleotide B species and polynucleotide B′ species pairs may have different lengths than each other. 
     
     
         50 . The composition of  claim 46 , wherein each of the polynucleotide B species and the polynucleotide B′ species are about 4 to about 20 nucleotides in length. 
     
     
         51 . The composition of  claim 46 , wherein there are 300 or fewer polynucleotide B species. 
     
     
         52 . The composition of  claim 46 , wherein the polynucleotide B species are annealed to the complementary polynucleotide B′ species and the polynucleotide A′ species does not anneal to the polynucleotide A species. 53 (Original) The composition of  claim 46 , wherein the composition is prepared for a sequencing reaction wherein the ratio of nucleic acid templates to polynucleotide B species is greater than 1,000 to 1. 
     
     
         54 . The composition of  claim 46 , wherein the first oligonucleotide and the second oligonucleotide are partially matched reverse complement pairs selected from SEQ ID NOs: 1-576. 
     
     
         55 . (canceled) 
     
     
         56 . A system for determining a sequence of nucleotides for one or more nucleic acid templates in a nucleic acid sample, comprising:
 one or more processors; and memory coupled to one or more processors, the memory encoded with a set of instructions configured to perform a process comprising the method steps of  claim 1 .   
     
     
         57 . (canceled) 
     
     
         58 . The composition of  claim 46 , wherein the partially double-stranded nonrandom oligonucleotide adapter species is a Y adapter or a hairpin adapter. 
     
     
         59 . The composition of  claim 46 , wherein the polynucleotide B species and the polynucleotide B′ species are non-degenerate. 
     
     
         60 . The composition of  claim 46 , wherein each of the first oligonucleotide species comprises a polynucleotide C species between polynucleotide A and the polynucleotide B species;
 each of the second oligonucleotide species comprises a polynucleotide C′ species between polynucleotide A′ and the polynucleotide B′ species;   each polynucleotide C′ species is the reverse complement of the polynucleotide C species; and   
       the polynucleotide C species anneal to complementary polynucleotide C′ species. 
     
     
         61 . The composition of  claim 46 , wherein each of the polynucleotide C species comprises the same nucleotide sequence or wherein the polynucleotide C species comprises at least two different nucleotide sequences. 
     
     
         62 . The composition of  claim 46 , wherein the nonrandom oligonucleotide adapter species comprise a blunt end. 
     
     
         63 . The composition of  claim 46 , wherein the double-stranded nonrandom oligonucleotide adapter species comprises a ligation linker. 
     
     
         64 . A kit comprising the composition of  claim 46 . 
     
     
         65 . The kit of  claim 64 , wherein the kit further comprises a list of the sequence information of B species and B′ species of the nonrandom oligonucleotide adapters. 
     
     
         65 . The kit of  claim 64 , further comprising reagents for treating the nucleic acid templates to generate blunt-ended nucleic acid templates. 
     
     
         66 . A method of determining a sequence of nucleotides for one or more nucleic acid templates in a nucleic acid sample, comprising contacting double-stranded nucleic acid templates of the nucleic acid sample with partially double-stranded nonrandom oligonucleotide adapter species in the composition of  claim 46  under ligation conditions, thereby generating adapter-ligated nucleic acid templates,
 amplifying the adapter-ligated nucleic acid templates, thereby generating amplicons; and 
 sequencing all or a portion of each amplicon, thereby determining a sequence of nucleotides for the one or more nucleic acid templates in the nucleic acid sample. 
 
     
     
         67 . A method of counting nucleic acid templates for a nucleic acid sample, comprising
 contacting double-stranded nucleic acid templates of the nucleic acid sample with partially double-stranded nonrandom oligonucleotide adapter species in the composition of  claim 46  under ligation conditions, thereby generating adapter-ligated nucleic acid templates:   amplifying the adapter-ligated nucleic acid templates, thereby generating amplicons;   identifying a set of amplicon duplicates, wherein the amplicon duplicates comprise amplified adapter-ligated nucleic acid templates comprising a polynucleotide B species at one end; and   determining the number of amplicon duplicates comprising the polynucleotide B species.   
     
     
         68 . The method of  claim 66 , wherein the double-stranded nucleic acid templates are double-stranded DNA templates or RNA templates. 
     
     
         69 . The method of  claim 67 , wherein the double-stranded nucleic acid templates are double-stranded DNA templates or RNA templates. 
     
     
         70 . The method of  claim 66 , wherein the double-stranded nucleic acid templates comprise a ligation linker. 
     
     
         71 . The method of  claim 67 , wherein the double-stranded nucleic acid templates comprise a ligation linker. 
     
     
         72 . The method of  claim 66 , wherein the ligation linker comprises at least one of a A-overhang, T-overhang, a CG-overhang, a blunt end, or any ligatable nucleic acid sequence. 
     
     
         73 . The method of  claim 67 , wherein the ligation linker comprises at least one of a A-overhang, T-overhang, a CG-overhang, a blunt end, or any nucleic acid sequence. 
     
     
         74 . The method of  claim 66 , wherein each of the adapter-ligated nucleic acid templates comprises a first nonrandom oligonucleotide adapter at a first end and a second nonrandom oligonucleotide adapter at a second end. 
     
     
         75 . The method of  claim 67 , wherein each of the adapter-ligated nucleic acid templates comprises a first nonrandom oligonucleotide adapter at a first end and a second nonrandom oligonucleotide adapter at a second end.