Methods and compositions for addressing inefficiencies in amplification reactions

Abstract

Methods and systems for decreasing amplification bias and primer-dimer formation in amplification reactions and for amplifying a plurality of target polynucleotides from a sample in a single reaction and for sequencing the target polynucleotides where samples can include forensic samples and where target polynucleotides can include identity- or ancestry-informative markers, short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). Methods of determining a nucleotide spacer sequence for disrupting primer dimer formation can include: receiving a set of primer sequences; determining a plurality of candidate spacers between an adapter sequence and a gene-specific portion of the primer sequence, the determined plurality of candidate spacers comprises sequences that disrupt stable interactions between sequences of the set of primer sequences; ranking candidate spacers that meet a predetermined threshold value of stable interactions in the extension sequences; and outputting a set of the ranked spacers that meet the predetermined threshold.

What is claimed is: 
     
         1 . An oligonucleotide composition, comprising a plurality of primers, each primer comprising a target nucleic acid specific sequence (TS) and wherein the plurality of primers comprises two or more quality control sequence (QCS) selected from the group consisting of
 a first QCS (QCS1), wherein each nucleic acid position is fully randomized,   a second QCS (QCS2), wherein one or more nucleic acid positions are partially randomized,   a third QCS (QCS3), wherein one or more nucleic acid positions are fixed,   a fourth QCS (QCS4), wherein all nucleic acid positions are fixed,   a fifth QCS (QCS5), wherein one or more nucleic acid positions are fully randomized and one or more nucleic acid positions are partially randomized,   a sixth QCS (QCS6), wherein one or more nucleic acid positions are fully randomized and one or more nucleic acid positions are fixed,   a seventh QCS (QCS7), wherein one or more nucleic acid positions are partially randomized and one or more nucleic acid position are fixed, and   an eighth QCS (QCS8), wherein one or more nucleic acid positions are fully randomized, one or more nucleic acid positions are partially randomized, and one or more nucleic acid positions are fixed.   
     
     
         2 . The oligonucleotide composition of  claim 1 , wherein the plurality of primers comprises 3, 4, 5, 6, 7, or 8 QCSs selected from the group consisting of QCS1, QCS2, QCS3, QCS4, QCS5, QCS6, QCS7, and QCS8. 
     
     
         3 . The oligonucleotide composition of  claim 2 , wherein the QCS of one or more primers is flanked by one or two extension sequences (ES). 
     
     
         4 . The oligonucleotide composition of  claim 3 , wherein one or more primers comprises an adaptor sequence (AS) on the 5′-end of the QCS. 
     
     
         5 . The oligonucleotide composition of  claim 4 , wherein one or more primers comprises an ES between the AS and the QCS (AES) or in between the QCS and the TS (TES). 
     
     
         6 . The oligonucleotide composition of  claim 3 , wherein the ES is a fixed sequence. 
     
     
         7 . The oligonucleotide composition of  claim 3 , wherein the ES comprises a sequence of between 1 and 10 bases. 
     
     
         8 . The oligonucleotide composition of  claim 7 , wherein the ES comprises a sequence of between 2 and 5 bases. 
     
     
         9 . The oligonucleotide composition of  claim 3 , wherein the QCS flanked by the ES is QCS1, QCS2, QCS3, QCS4, QCS5, QCS6, QCS7, or QCS8. 
     
     
         10 . The oligonucleotide composition of  claim 1 , wherein QCS1, QCS2, QCS3, QCS4, QCS5, QCS6, QCS7, or QCS8 comprises a sequence of 3-12 bases. 
     
     
         11 . The oligonucleotide composition of  claim 1 , wherein the plurality of primers comprises between about 4 primers and about 5000 primers. 
     
     
         12 . The oligonucleotide composition of  claim 11 , wherein the plurality of primers comprises between about 4 primers and about 550 primers. 
     
     
         13 . The oligonucleotide composition of  claim 11 , wherein at least one forward primer of the plurality of primer pairs comprises a QCS selected from the group consisting of QCS1, QCS2, QCS3, QCS4, QCS5, QCS6, QCS7, and QCS8, and wherein at least one reverse primer of the plurality of primer pairs comprises a QCS selected from the group consisting of QCS1, QCS2, QCS3, QCS4, QCS5, QCS6, QCS7, and QCS8. 
     
     
         14 . The oligonucleotide composition of  claim 1 , wherein the TS of one or more primers is complementary to a region flanking a short tandem repeat (STR) region. 
     
     
         15 . The oligonucleotide composition of  claim 1 , wherein the plurality of primers comprises one or more primers selected from the group consisting of D165359, D61043, DYS 570 , D195433, PentaD, DYS 576 , AmelPP, DXS10135, D135317, DYS 389 , D20S 482 , DXS10074, rs1805009, rs10776839, rs2831700, rs1042602, rs1058083, DYS 392 , D22S1045, DYS19, DYS 456 , DYS 439 , and DYS635. 
     
     
         16 . A method for assembling an oligonucleotide composition provided herein, comprising:
 a) providing an initial primer pool including a plurality of primers wherein each primer includes a target sequence;   b) amplifying target polynucleotides from a sample using the initial primer pool;   c) identifying a subgroup of primers in the initial primer pool wherein the products of amplification result in an inaccurate allelic ratio or increased formation of primer dimers;   d) modifying one or more primers in the subgroup of primers wherein modifying comprises:
 i) modifying one or more primers to include a quality control sequence (QCS); 
 ii) modifying one or more QCS to include one or more extension sequences (ES); 
   e) repeating steps b-d with modified primers until the products of amplification result in an accurate allelic ratio or decreased formation of primer dimers;   thereby producing an optimized primer pool.   
     
     
         17 . The method of  claim 16 , wherein a QCS used to modify a primer in the initial primer pool is selected from the group consisting of QCS1, QCS2, QCS3, QCS4, QCS5, QCS6, QCS7 and QCS8. 
     
     
         18 . A computer-implemented method of determining a nucleotide spacer sequence for disrupting primer dimer formation, comprising:
 receiving a set of primer sequences;   determining, using at least one microprocessor, a plurality of candidate spacers between an adapter sequence and a gene-specific portion of the primer sequence, the determined plurality of candidate spacers comprises sequences that disrupt stable interactions between sequences of the set of primer sequences;   computing, using at least one microprocessor, a set of candidate spacers that meet a predetermined threshold value of stable interactions in the extension sequences; and   outputting a set of the ranked spacers that meet the predetermined threshold.   
     
     
         19 . The method of  claim 18 , wherein the plurality of spacers is in between a molecular tag and one of the adapter sequence and the gene-specific portion of the primer sequence. 
     
     
         20 . The method of  claim 19 , wherein determining spacer sequences comprises determining a gene-specific side sequence that flanks a first side of the molecular tag. 
     
     
         21 . The method of  claim 20 , wherein determining spacer sequences further comprises determining an adapter side sequence that flanks a second side of the molecular tag. 
     
     
         22 . The method of  claim 21 , wherein determining the candidate spacers includes:
 determining, using at least one microprocessor, taboo seeds based on sequences that complement the primer; and   removing sequences that include the taboo seeds from the candidate spacers.   
     
     
         23 . The method of  claim 22 , wherein computing the set of candidate spacers includes updating the taboo seed flank with adjacent base pair sequences from the outer genomic flank. 
     
     
         24 . The method of  claim 23 , wherein computing the set of candidate spacers is based on alignment edit distances between the candidate spacers and the taboo sequences. 
     
     
         25 . The method of  claim 18 , wherein computing the set of candidate spacers comprises checking whether a portion of the spacer sequences matches oppositely reversed complimentary primers. 
     
     
         26 . The method of  claim 25 , further comprising designing the molecular tag to be less than completely random depending on the outputting of the set of the computed spacers.