Novel primers and uses thereof

Abstract

A composition comprising a primer, the primer being: (a) a circle-forming primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the stem-forming segment is hybridizable to a portion of the target-specific segment to form a stem structure, or (b) a split primer comprising a first target-specific segment, a second target-specific segment, and an adaptor segment between the first and second target-specific segments, or (c) a split-circle-forming primer comprising a first target-specific segment, a second target-specific segment, and a stem-forming segment between the first and second target-specific segments, and an adaptor segment, or comprising a first adaptor segment, a second adaptor segment, and a stem-forming segment between the first and second adaptor segments, and a target-specific segment. Also disclosed is a method of amplifying a target locus of interest from a template DNA, the method comprising at least two pre-amplification cycles using a circle-forming primer, a split primer, and / or a split-circle-forming primer, wherein each amplification cycle comprises annealing the primer to the template DNA or a pre-amplification product thereof and extending the annealed primer. Also disclosed is a kit for amplifying a target locus of interest, comprising a circle-forming primer, a split primer, and / or a split-circle-forming primer.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 617,066, filed January 12, 2018, which is incorporated by reference in its entirety.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 27, 2018, is named N_022_WO_01_SL.txt and is 3,844 bytes in size. BACKGROUND

[0005] Molecular barcodes or index sequences have been used in next generation sequencing to reduce replication-induced quantification bias by labeling each nucleic acid fragment with a molecular barcode or index sequence. Sequence reads with different molecular barcodes or index sequences represent different original nucleic acid molecules. By referencing the molecular barcode or index sequence, PCR products such as sequence changes produced by polymerase errors that are not present in the original nucleic acid molecule can be identified and separated from true variants / mutations present in the original nucleic acid molecule.

[0006] However, to apply molecular barcodes or index sequences in highly multiplexed PCR, the formation of primer dimers needs to be suppressed, barcode resampling needs to be avoided, and non-specific primer binding and primer concatenation formation needs to be reduced. SUMMARY

[0007] The present invention relates to compositions, methods, and kits for amplifying nucleic acids. In a first aspect, the invention described herein relates to a composition comprising a primer that is: (a) a circle-forming primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the stem-forming segment is hybridizable to a portion of the target-specific segment to form a stem structure, or (b) a split primer comprising a first target-specific segment, a second target-specific segment, and an adaptor segment positioned between the first target-specific segment and the second target-specific segment, or (c) a split-circle-forming primer comprising a first target-specific segment, a second target-specific segment, and a stem-forming segment positioned between the first target-specific segment and the second target-specific segment, and an adaptor segment, or comprising a first adaptor segment, a second adaptor segment, and a stem-forming segment positioned between the first adaptor segment and the second adaptor segment, and a target-specific segment.

[0008] In a second aspect, the invention described herein relates to a method of amplifying a target locus of interest from a template DNA, the method comprising at least two pre-amplification cycles using primers that are: (a) a loopable primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the stem-forming segment is hybridizable to a portion of the target-specific segment to form a stem structure, or (b) a split primer comprising a first target-specific segment, a second target-specific segment, and an adaptor segment located between the first and second target-specific segments, or (c) a split-loopable primer comprising a first target-specific segment, a second target-specific segment, and a stem-forming segment located between the first and second target-specific segments, and an adaptor segment, or comprising a first adaptor segment, a second adaptor segment, and a stem-forming segment located between the first and second adaptor segments, and a target-specific segment; wherein each pre-amplification cycle comprises annealing the primers to the template DNA or a pre-amplification product thereof and extending the annealed primers.

[0009] In a third aspect, the invention described herein relates to a kit for amplifying a target locus of interest, comprising primers that are: (a) a loopable primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the stem-forming segment is hybridizable to a portion of the target-specific segment to form a stem structure, or (b) a split primer comprising a first target-specific segment, a second target-specific segment, and an adaptor segment located between the first and second target-specific segments, or (c) a split-loopable primer comprising a first target-specific segment, a second target-specific segment, and a stem-forming segment located between the first and second target-specific segments, and an adaptor segment, or comprising a first adaptor segment, a second adaptor segment, and a stem-forming segment located between the first and second adaptor segments, and a target-specific segment.

[0010] In a fourth aspect, the invention described herein relates to a method of determining variant copy number of a target locus of interest, the method comprising: pre-amplifying the target locus of interest from template DNA using at least two pre-amplification cycles performed using: (a) one or more loopable primers each comprising a target-specific segment, an adaptor segment, a molecular index segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the adaptor segment and the molecular index segment and the 5' portion of the target-specific segment, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the molecular index segment comprises a molecular index sequence, (b) one or more split-loopable primers each comprising a first target-specific segment, a second target-specific segment, a stem-forming segment located between the first target-specific segment and the second target-specific segment, a molecular index segment, and an adaptor segment, wherein the stem-forming segment is hybridizable to a portion of the second target-specific segment to form a stem structure and a loop comprising the adaptor segment and the molecular index segment, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the molecular index segment comprises a molecular index sequence, or (c) one or more split-loopable primers each comprising a first adaptor segment, a second adaptor segment, a stem-forming segment located between the first adaptor segment and the second adaptor segment, a molecular index segment, and a target-specific segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the second adaptor segment and the molecular index segment and the 5' portion of the target-specific segment, wherein the first and / or second adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the molecular index segment comprises a molecular index sequence; amplifying the pre-amplification product using one or more PCR primers hybridizable to the universal adaptor sequence; and sequencing the amplification product to determine variant copy number of the target locus of interest using the molecular index sequence.

[0011] In a fifth aspect, the invention described herein relates to a method of determining a fetal aneuploidy, the method comprising: pre-amplifying a plurality of target loci of one or more chromosomes from cell-free DNA isolated from a maternal blood sample using at least two pre-amplification cycles performed using: (a) a plurality of loopable primers each comprising a target-specific segment, an adaptor segment, a molecular index segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the adaptor segment and the molecular index segment and the 5' portion of the target-specific segment, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the molecular index segment comprises a molecular index sequence, (b) a plurality of split-loopable primers each comprising a first target-specific segment, a second target-specific segment, a stem-forming segment located between the first target-specific segment and the second target-specific segment, a molecular index segment, and an adaptor segment, wherein the stem-forming segment is hybridizable to a portion of the second target-specific segment to form a stem structure and a loop comprising the adaptor segment and the molecular index segment, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the molecular index segment comprises a molecular index sequence, or (c) a plurality of split-loopable primers each comprising a first adaptor segment, a second adaptor segment, a stem-forming segment located between the first adaptor segment and the second adaptor segment, a molecular index segment, and a target-specific segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the second adaptor segment and the molecular index segment and the 5' portion of the target-specific segment, wherein the first and / or second adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the molecular index segment comprises a molecular index sequence; amplifying the pre-amplification products using one or more PCR primers hybridizable to the universal adaptor sequence; and sequencing the amplification products to determine a fetal aneuploidy using the molecular index sequences.

[0012] In a sixth aspect, the invention described herein relates to a method of multiplexed amplification, the method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles performed using: (a) at least a first and a second loopable primers each comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the adaptor segment and the 5' portion of the target-specific segment, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, (b) at least a first and a second split-loopable primers each comprising a first target-specific segment, a second target-specific segment, a stem-forming segment located between the first and second target-specific segments, and an adaptor segment, wherein the stem-forming segment is hybridizable to a portion of the second target-specific segment to form a stem structure and a loop comprising the adaptor segment, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, or (c) at least a first and a second split-loopable primers each comprising a first adaptor segment, a second adaptor segment, a stem-forming segment located between the first and second adaptor segments, and a target-specific segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the second adaptor segment and the 5' portion of the target-specific segment, wherein the first and / or second adaptor segments comprise a universal adaptor sequence for PCR amplification; and wherein the first and second primers comprise the complement of their target-specific segments and are capable of forming primer dimers in the absence of the stem-forming segment protection; and amplifying the pre-amplification products using one or more PCR primers hybridizable to the universal adaptor sequences. Preventing primer dimer formation is particularly useful for PCR tiling (e.g., amplifying overlapping or tiled target sequences in a single multiplexed PCR reaction).

[0013] In a seventh aspect, the invention described herein relates to a method of allele-specific amplification, the method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles performed using: (a) a loopable primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the adaptor segment and the 5' portion of the target-specific segment, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the loopable primer comprises an SNV or SNP at the 5' portion or the 3' portion of the target-specific segment, (b) a split-loopable primer comprising a first target-specific segment, a second target-specific segment, a stem-forming segment located between the first target-specific segment and the second target-specific segment, and an adaptor segment, wherein the stem-forming segment is hybridizable to a portion of the second target-specific segment to form a stem structure and a loop comprising the adaptor segment, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the split-loopable primer comprises an SNV or SNP at the target-specific segment, or (c) a split-loopable primer comprising a first adaptor segment, a second adaptor segment, a stem-forming segment located between the first adaptor segment and the second adaptor segment, and a target-specific segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the second adaptor segment and the 5' portion of the target-specific segment, wherein the first and / or second adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the split-loopable primer comprises an SNV or SNP allele at the 3' portion of the target-specific segment; and amplifying the pre-amplification product using one or more PCR primers hybridizable to the universal adaptor sequence.

[0014] In an eighth aspect, the invention described herein relates to an allele-specific quantitative PCR (qPCR) method, the method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles performed with: (a) at least a first and a second loopable primer, each comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the adaptor segment and the 5' portion of the target-specific segment, wherein the adaptor segment of the first loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adaptor segment of the second loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' portion or the 3' portion of the target-specific segment of the first loopable primer comprises a first SNV or SNP allele, and wherein the 5' portion or the 3' portion of the target-specific segment of the second loopable primer comprises a second SNV or SNP allele, (b) at least a first and a second split-loopable primer, each comprising a first target-specific segment, a second target-specific segment, a stem-forming segment located between the first and second target-specific segments, and an adaptor segment, wherein the stem-forming segment is hybridizable to a portion of the second target-specific segment to form a stem structure and a loop comprising the adaptor segment, wherein the adaptor segment of the first split-loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adaptor segment of the second split-loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the target-specific segment of the first split-loopable primer comprises a first SNV or SNP allele, and wherein the target-specific segment of the second split-loopable primer comprises a second SNV or SNP allele, or (c) at least a first and a second split-loopable primer, each comprising a first adaptor segment, a second adaptor segment, a stem-forming segment located between the first and second adaptor segments, and a target-specific segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the second adaptor segment and the 5' portion of the target-specific segment, wherein the adaptor segment of the first split-loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adaptor segment of the second split-loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' portion or the 3' portion of the target-specific segment of the first split-loopable primer comprises a first SNV or SNP allele, and wherein the 5' portion or the 3' portion of the target-specific segment of the second split-loopable primer comprises a second SNV or SNP allele, and performing at least one qPCR cycle using at least one of the following: (i) a first fluorescent probe capable of binding to the first probe-specific sequence of the first loopable primer or the first split-loopable primer, and (ii) a second fluorescent probe capable of binding to the second probe-specific sequence of the second loopable primer or the second split-loopable primer.The first and / or second adaptor segment of the first split-cyclable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the first and / or second adaptor segment of the second split-cyclable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' portion or 3' portion of the target-specific segment of the first split-cyclable primer comprises a first SNV or SNP allele, and wherein the 5' portion or 3' portion of the target-specific segment of the second split-cyclable primer comprises a second SNV or SNP allele; amplifying the pre-amplification product using one or more PCR primers hybridizable to the universal adaptor sequence in the presence of the first fluorescent probe and the second fluorescent probe; and detecting real-time fluorescent signal intensity from the first fluorescent probe and the second fluorescent probe. Alternatively, the method for allele-specific qPCR does not require a pre-amplification step, but comprises amplifying one or more target loci of interest from template DNA using the primers of (a), (b), or (c) in the presence of the first fluorescent probe and the second fluorescent probe; and detecting real-time fluorescent signal intensity from the first fluorescent probe and the second fluorescent probe.

[0015] In a ninth aspect, the invention described herein relates to an allele-specific digital PCR (dPCR) method, the method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles performed with: (a) at least a first loopable primer and a second loopable primer, each comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the adaptor segment and the 5' portion of the target-specific segment, wherein the adaptor segment of the first loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adaptor segment of the second loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' portion or the 3' portion of the target-specific segment of the first loopable primer comprises a first SNV or SNP allele, and, wherein the 5' portion or the 3' portion of the target-specific segment of the second loopable primer comprises a second SNV or SNP allele, (b) at least a first split-loopable primer and a second split-loopable primer, each comprising a first target-specific segment, a second target-specific segment, a stem-forming segment located between the first target-specific segment and the second target-specific segment, and an adaptor segment, wherein the stem-forming segment is hybridizable to a portion of the second target-specific segment to form a stem structure and a loop comprising the adaptor segment, wherein the adaptor segment of the first split-loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adaptor segment of the second split-loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the target-specific segment of the first split-loopable primer comprises a first SNV or SNP allele, and, wherein the target-specific segment of the second split-loopable primer comprises a second SNV or SNP allele, or (c) at least a first split-loopable primer and a second split-loopable primer, each comprising a first adaptor segment, a second adaptor segment, a stem-forming segment located between the first adaptor segment and the second adaptor segment, and a target-specific segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure and a loop comprising the second adaptor segment and the 5' portion of the target-specific segment, wherein the adaptor segment of the first split-loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adaptor segment of the second split-loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' portion or the 3' portion of the target-specific segment of the first split-loopable primer comprises a first SNV or SNP allele, and, wherein the 5' portion or the 3' portion of the target-specific segment of the second split-loopable primer comprises a second SNV or SNP allele, and performing at least one dPCR cycle on the pre-amplified template DNA.the first and / or second adaptor segment of the first split-circularizable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the first and / or second adaptor segment of the second split-circularizable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' portion or 3' portion of the target-specific segment of the first split-circularizable primer comprises a first SNV or SNP allele, and wherein the 5' portion or 3' portion of the target-specific segment of the second split-circularizable primer comprises a second SNV or SNP allele; dividing the pre-amplification product into a plurality of reaction volumes; amplifying the pre-amplification product in each reaction volume using one or more PCR primers that can hybridize to the universal adaptor sequence in the presence of the first fluorescent probe and the second fluorescent probe; and detecting the presence or absence of fluorescent signal from the first fluorescent probe and the second fluorescent probe. Alternatively, the method for allele-specific dPCR does not require a pre-amplification step, but instead comprises dividing the sample into a plurality of reaction volumes; amplifying one or more target loci of interest from the template DNA in each reaction volume using the primers of (a), (b), or (c) in the presence of a first fluorescent probe and a second fluorescent probe; and detecting the presence or absence of fluorescent signal from the first fluorescent probe and the second fluorescent probe.

[0016] These and other features, together with the organization and operation of the application, will be better understood from the following detailed description when considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Some embodiments of primers described herein are shown (Schemes A-C and control scheme).

[0018] Figure 2 An embodiment of a circularizable primer is shown, in which the 3' end portion of the target-specific segment is configured to form a stem structure with a complementary sequence.

[0019] Figure 3 An embodiment of a circularizable primer is shown, in which the 5' end portion of the target-specific segment is configured to form a stem structure with a complementary sequence.

[0020] Figure 4 An embodiment of a split primer is shown, in which the target-specific segment is split into a 5' end portion and a 3' end portion separated by an adaptor sequence.

[0021] Figure 5 A workflow showing an exemplary amplification process (including 2 pre-amplification cycles) using primers described herein is shown.

[0022] Figure 6 Quasi-target rates are shown for an exemplary amplification process using the primers described herein (including 2 pre-amplification cycles).

[0023] Figure 7 Workflows are shown for exemplary amplification processes using the primers described herein (including 3 or 10 pre-amplification cycles).

[0024] Figure 8 Quasi-target rates are shown for an exemplary amplification process using the primers described herein (including 3 or 10 pre-amplification cycles).

[0025] Figure 9 Consistent MIT counts between replicate samples are shown (Protocol A, 2 pre-amplification cycles).

[0026] Figure 10 Primer and product sequences are shown according to one embodiment of a loopable primer, which includes 2 mismatched nts in the loopable primer.

[0027] Figure 11 Primer and product sequences are shown according to one embodiment of a loopable primer.

[0028] Figure 12 Primer and product sequences are shown according to one embodiment of a split primer.

[0029] Figure 13 Some embodiments of split-loopable primers described herein are shown (Protocols D-E).

[0030] Figure 14 Consistent MIT counts between replicate samples are shown in a highly multiplexed PCR (Protocol A, 2 pre-amplification cycles, workflow as shown in Figure 5 Quasi-target rates for this exemplary amplification process were 83%. DETAILED DESCRIPTION DETAILED DESCRIPTION

[0031] Reference will now be made in detail to some specific embodiments of the application, as the inventors have conceived the application to achieve. Certain examples of these specific instances are shown in the accompanying drawings. While the application is described in conjunction with these specific instances, it will be understood that it is not intended to limit the application to the described instances. On the contrary, it is intended to cover alternatives, modifications, and equivalents, which can be included within the spirit and scope of the application as defined by the appended claims.

[0032] In the following description, numerous specific details are set forth to provide a thorough understanding of the application. Examples of specific embodiments of the application can be practiced without some or all of these specific details.

[0033] For clarity, various techniques and mechanisms of the present application are sometimes described in singular form. However, it should be noted that some embodiments include multiple iterations of a technique or multiple examples of a mechanism, unless otherwise indicated.

[0034] The disclosures of the following patent applications are incorporated herein by reference: International Patent Application No. PCT / US2006 / 045281, entitled "Systems and Methods for Cleaning Noisy Genetic Data and Using the Data for Prediction"; International Patent Application No. PCT / US2008 / 003547, entitled "Systems and Methods for Cleaning Noisy Genetic Data and Determining Chromosome Copy Number"; International Patent Application No. PCT / US2009 / 034506, entitled "Cell Genotyping Methods"; International Patent Application No. PCT / US2009 / 045335, entitled "Embryo Characterization and Comparison Methods"; International Patent Application No. PCT / US2009 / 052730, entitled "Methods for Allele Identification and Ploidy Identification"; International Patent Application No. PCT / US2010 / 050824, entitled "Methods for Non-Invasive Prenatal Ploidy Identification"; International Patent Application No. PCT / US2011 / 037018, entitled "Methods for Non-Invasive Prenatal Ploidy Identification"; International Patent Application No. PCT / US2011 / 061506, entitled "Methods for Non-Invasive Prenatal Ploidy Identification"; International Patent Application No. PCT / US2011 / 066938, entitled "Non-Invasive Prenatal Paternity Methods"; International Patent Application No. PCT / US2012 / 066339, entitled "Highly Multiplexed PCR Methods and Compositions"; International Patent Application No. PCT / US2013 / 055205, entitled "Methods and Compositions for Reducing Contamination of Genetic Libraries"; International Patent Application No. PCT / US2013 / 057924, entitled "Methods for Increasing Fetal Fraction in Maternal Blood"; International Patent Application No. PCT / US2014 / 051926, entitled "Methods Using Low Fetal Fraction Detection"; International Patent Application No. PCT / US2014 / 057843, entitled "Prenatal Diagnostic Rest Criteria"; International Patent Application No. PCT / US2015 / 026957, entitled "Detecting Mutations and Ploidy of Chromosomal Segments"; International Patent Application No. PCT / US2016 / 031686, entitled "Methods and Compositions for Determining Ploidy"; and U.S. Patent Application No. 15 / 372,279, entitled "Compositions and Methods for Nucleic Acid Identification".

[0035] Loopable primer

[0036] Many embodiments of the application described herein relate to loopable primers that include a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the stem-forming segment can hybridize to a portion of the target-specific segment to form a stem structure, and wherein the target-specific segment can hybridize to a target sequence of a template DNA for amplification.

[0037] In some embodiments, the adaptor segment is located between the target-specific segment and the stem-forming segment, wherein hybridization between the stem-forming segment and a portion of the target-specific segment forms a loop that includes the adaptor segment. For example, the adaptor segment can be located 5' to the target-specific segment and 3' to the stem-forming segment. In some embodiments, the adaptor segment includes a universal adaptor sequence for PCR amplification and / or sequencing.

[0038] In some embodiments, the loopable primer further includes a molecular index segment that includes a molecular index sequence. Molecular index sequences, molecular index tags (MITs), or unique identifier (UID) sequences are described in Kinde et al., PNAS 108(23):9530-9535 (2011), and U.S. Patent Application No. 15 / 372,279, entitled “Compositions and Methods for Nucleic Acid Molecular Identification,” each of which is incorporated by reference herein in its entirety. In some embodiments, each molecular index sequence is about 1-20 bp, or about 2-15 bp, or about 3-10 bp, or about 4-8 bp in length. When both an upstream loopable primer and a downstream loopable primer according to the application described herein are used to amplify a target locus of interest, the amplification product can include two molecular index sequences, the combination of which can provide more accurate molecular counts compared to using a single molecular index sequence. In one embodiment, the molecular index sequence on each loopable primer is a unique molecular index sequence. In another embodiment, the combination of molecular index sequences on each pair of upstream and downstream loopable primers is unique.

[0039] For example, the molecular index segment can be located between the target-specific segment and the adaptor segment. For example, the molecular index segment is located 5' to the target-specific segment and 3' to the adaptor segment. In some embodiments, hybridization between the stem-forming segment and a portion of the target-specific segment forms a loop that includes the adaptor segment and the molecular index segment.

[0040] In some embodiments, the loopable primers described herein do not include primers in which the target-specific segment does not form part of a stem structure.

[0041] Since the circularizable primers described herein can hide / protect at least a portion of the universal adaptor sequence, molecular index sequence, and target-specific sequence, they can significantly improve assay specificity in highly complexed PCR by inhibiting primer dimer and nonspecific binding.

[0042] Option A: Figure 2 A scheme for a circular primer is shown, wherein the target-specific region comprises a 5' portion and a 3' portion, wherein a stem-forming region can hybridize with the 3' portion of the target-specific region to form a stem structure. When the 3' portion of the target-specific region is protected within the stem structure, the 5' portion of the target-specific region can be used to initiate target hybridization.

[0043] In some embodiments, hybridization between the stem-forming segment and the 3' portion of the target-specific segment forms a loop, the loop comprising an adaptor segment and a 5' portion of the target-specific segment. The stem-loop is designed to protect the molecular index sequence and adaptor sequence from spurious interactions. The 3' end stem also prevents primer non-target-specific extension (i.e., primer dimerization).

[0044] In some embodiments, the circulating primer further includes one or more mismatched nucleotides at the 3' end of the target-specific region, the mismatched nucleotides being non-hybridizable to the stem-forming region. In some embodiments, the circulating primer includes 1, 2, 3, 4, or 5 mismatched nucleotides at the 3' end to prevent A-tailing. Optionally or additionally, the 5' end of the circulating primer may include one or more mismatched nucleotides that are non-hybridizable to the target-specific region. In some embodiments, the circulating primer includes 1, 2, 3, 4, or 5 mismatched nucleotides at the 5' end.

[0045] like Figure 10 As shown, a preferred embodiment of the circulant primer according to scheme A includes one or more mismatched nucleotides, a stem-forming region, an adaptor region, a molecular indexing region, and a target-specific region from the 5' to 3' direction, wherein the stem-forming region is the downstream complement of the 3' portion of the target-specific region.

[0046] In some embodiments, the size of the stem structure formed between the 3' portion of the stem-forming region and the target-specific region is about 5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20 bp.

[0047] In some embodiments, the circularizable primers according to Scheme A have preferred annealing and melting temperatures for PCR reactions, wherein the 3' portion of the stem-forming region and the target-specific region forms a stem structure at or below the preferred annealing temperature, but does not form a stem structure at or above the melting temperature. In some instances, preferred annealing temperatures are 60°C or below, 59°C or below, 58°C or below, 57°C or below, 56°C or below, 55°C or below, 54°C or below, 53°C or below, 52°C or below, 51°C or below, or 50°C or below. In some embodiments, melting temperatures are 60°C or above, 61°C or above, 62°C or above, 63°C or above, 64°C or above, 65°C or above, 66°C or above, 67°C or above, 68°C or above, 69°C or above, or 70°C or above. In some instances, extreme annealing temperatures, such as 30°C to 80°C, may be useful.

[0048] Option B: Figure 3 Another scheme for a loopable primer is shown, wherein the target-specific region comprises a 5' portion and a 3' portion, wherein a stem-forming region can hybridize with the 5' portion of the target-specific region to form a stem structure. When the 5' portion of the target-specific region is protected within the stem structure, the 3' portion of the target-specific region can be used to initiate the target.

[0049] In some embodiments, a loop is formed by hybridization between the stem-forming segment and the 5' portion of the target-specific segment. The loop includes an adaptor segment but excludes the 3' terminal portion of the target-specific segment. The stem loop is designed to protect the molecular index sequence and adaptor sequence from spurious interactions.

[0050] In some embodiments, the circulating primer further comprises one or more G / C nucleotides located on the 5' side of the target-specific region for stabilizing the stem structure together with one or more complementary G / C nucleotides located on the 3' side of the stem-forming region. In some embodiments, the circulating primer comprises 1, 2, 3, 4, or 5 G / C nucleotides on the 5' side of the target-specific region (i.e., the neck of the stem loop).

[0051] like Figure 11 As shown, a preferred embodiment of the circulant primer according to scheme B includes, from the 5' to 3' direction, a stem-forming region, one or more G / C nucleotides, an adaptor region, a molecular index region, one or more G / C nucleotides, and a target-specific region, wherein the stem-forming region is the downstream complement of the 5' portion of the target-specific region.

[0052] In some embodiments, the stem structure formed between the stem-forming segment and the 5' portion of the target-specific segment is about 5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20 bp in size.

[0053] In some embodiments, the loopable primer according to Scheme B has a preferred annealing temperature and a melting temperature for a PCR reaction, wherein the stem-forming segment and the 5' portion of the target-specific segment form a stem structure at or below the preferred annealing temperature and do not form a stem structure at or above the melting temperature. In some examples, the preferred annealing temperature is 60°C or below, or 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, or 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below. In some embodiments, the melting temperature is 60°C or above, or 61°C or above, or 62°C or above, or 63°C or above, or 64°C or above, or 65°C or above, or 66°C or above, or 67°C or above, or 68°C or above, or 69°C or above, or 70°C or above. In some examples, extreme annealing temperatures can be useful, such as 30°C to 80°C.

[0054] Split primers

[0055] Many embodiments of the application described herein relate to split primers, which include a first target-specific segment, a second target-specific segment, and an adaptor segment located between the first target-specific segment and the second target-specific segment, wherein the target-specific segments are hybridizable to a target sequence of a template DNA intended for amplification.

[0056] In some embodiments, the split primers further include a molecular index segment, which includes a molecular index sequence. For example, the molecular index segment can be located between the adaptor segment and one of the target-specific segments. For example, the molecular index segment can be located 3' to the adaptor segment. In some embodiments, each molecular index sequence is about 1-20 bp, or about 2-15 bp, or about 3-10 bp, or about 4-8 bp in length. When both upstream and downstream split primers according to the application described herein are used to amplify a target locus of interest, the amplification product can include two molecular index sequences, which in combination can provide more accurate molecular counting compared to using a single molecular index sequence. In one embodiment, the molecular index sequence on each split primer is a unique molecular index sequence. In another embodiment, the combination of molecular index sequences on each pair of upstream and downstream split primers is unique.

[0057] In some embodiments, the adaptor segment includes a universal adaptor sequence for PCR amplification and / or sequencing.

[0058] Scheme C: Figure 4 One scheme for splitting the primer is shown, where an adapter segment is located between a first target-specific segment and a second target-specific segment. Both the first and second target-specific segments can be used to hybridize to a target sequence.

[0059] In other words, the target-specific segments are split into two parts, and a universal adapter sequence is located between the two parts. After binding of both ends of the primer to the target sequence, the molecular index sequence and the adapter sequence are protected. Split primers can have an advantage in reducing sequencing distance.

[0060] As shown in Scheme C, a preferred embodiment of a split primer according to Scheme C includes, in the 5' to 3' direction, a first target-specific segment, an adapter segment, a molecular index segment, a second target-specific segment. Figure 12 Split-circularizable primers

[0061] Many embodiments of the application described herein relate to split-circularizable primers that (a) include a first target-specific segment, a second target-specific segment, and a stem-forming segment located between the first and second target-specific segments, and an adapter segment, or (b) include a first adapter segment, a second adapter segment, and a stem-forming segment located between the first and second adapter segments, and a target-specific segment; and wherein the (first and / or second) target-specific segment(s) can hybridize to a target sequence of a template DNA intended for amplification.

[0062] In some embodiments, the split-circularizable primers further include a molecular index segment that includes a molecular index sequence. For example, the molecular index segment can be located between the adapter segment and the (second) target-specific segment. For example, the molecular index segment can be located 3' to the (second) adapter segment. In some embodiments, each molecular index sequence is about 1-20 bp, or about 2-15 bp, or about 3-10 bp, or about 4-8 bp in length. When both an upstream split-circularizable primer and a downstream split-circularizable primer according to the application described herein are used to amplify a target locus of interest, the amplification product can include two molecular index sequences, the combination of which can provide more accurate molecular counting compared to using a single molecular index sequence. In one embodiment, the molecular index sequence on each split-circularizable primer is a unique molecular index sequence. In another embodiment, the combination of molecular index sequences on each pair of upstream and downstream split-circularizable primers is unique.

[0063]

[0064] ​In some embodiments, the (first and / or second) adaptor segments include universal adaptor sequences for PCR amplification and / or sequencing.

[0065] Option D: Figure 13 A scheme for a segmentation-circular primer is shown, comprising a first target-specific region, a second target-specific region, a stem-forming region located between the first and second target-specific regions, and a connective region, wherein the stem-forming region can hybridize with the second target-specific region to form a stem structure. When the second target-specific region is protected within the stem structure, the first target-specific region can be used to initiate target hybridization.

[0066] In some embodiments, the hybridization between the stem-forming segment and the second target-specific segment forms a loop, the loop comprising the adaptor segment and the molecular index sequence. The stem loop is designed to protect the molecular index sequence and the adaptor sequence from spurious interactions. The 3' end stem also prevents primer non-target-specific extension (i.e., primer dimerization).

[0067] In some embodiments, the split-circular primer further includes one or more mismatched nucleotides at the 3' end of the second target-specific region, the mismatched nucleotides being non-hybridizable with the stem-forming region. In some embodiments, the split-circular primer includes 1, 2, 3, 4, or 5 mismatched nucleotides at the 3' end to prevent A-tailing. Optionally or additionally, the 5' end of the stem-forming region may include one or more mismatched nucleotides that are non-hybridizable with the second target-specific region. In some embodiments, the split-circular primer includes 1, 2, 3, 4, or 5 mismatched nucleotides at the 5' end of the stem-forming region.

[0068] like Figure 13 As shown, a preferred embodiment of the segmentation-circular primer according to scheme D includes, from the 5' to 3' direction, a first target-specific segment, one or more mismatched nucleotides, a stem-forming segment, an adaptor segment, a molecular index segment, and a second target-specific segment, wherein the stem-forming segment is a downstream complement of a portion of the second target-specific segment.

[0069] In some embodiments, the size of the stem structure formed between the stem-forming region and the second target-specific region is about 5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20 bp.

[0070] In some embodiments, the first target-specific segment is longer than the second target-specific segment. In some embodiments, the second target-specific segment is longer than the first target-specific segment.

[0071] In some embodiments, at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the second target-specific segment can hybridize to the stem-forming region and be capable of forming a stem.

[0072] In some embodiments, the split-circularizable primer according to Scheme D has a preferred annealing temperature and a melting temperature for a PCR reaction, wherein the stem-forming segment and the second target-specific segment form a stem structure at or below the preferred annealing temperature and do not form a stem structure at or above the melting temperature. In some examples, the preferred annealing temperature is 60°C or below, or 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, or 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below. In some embodiments, the melting temperature is 60°C or above, or 61°C or above, or 62°C or above, or 63°C or above, or 64°C or above, or 65°C or above, or 66°C or above, or 67°C or above, or 68°C or above, or 69°C or above, or 70°C or above. In some examples, an extreme annealing temperature can be useful, such as 30°C to 80°C.

[0073] Scheme E: Figure 13 Another scheme for a split-circularizable primer is shown, including a first adaptor segment, a second adaptor segment, and a stem-forming segment between the first adaptor segment and the second adaptor segment, and a target-specific segment, which includes a 5' portion and a 3' portion, wherein the stem-forming segment can hybridize to the 3' portion of the target-specific segment to form a stem structure. When the 3' portion of the target-specific segment is protected in the stem structure, the 5' portion of the target-specific segment can be used to initiate target hybridization.

[0074] In some embodiments, the hybridization between the stem-forming segment and the 3' portion of the target-specific segment forms a loop, which includes the second adaptor segment, the molecular index sequence, and the 5' portion of the target-specific segment. The stem loop is intended to protect the molecular index sequence and the second adaptor sequence from pseudo-interactions. The stem at the 3' end also prevents primer non-target-specific extension (i.e., primer dimer).

[0075] In some embodiments, the split-circular primer further includes one or more mismatched nucleotides at the 3' end of the target-specific region, the mismatched nucleotides being non-hybridizable with the stem-forming region. In some embodiments, the split-circular primer includes 1, 2, 3, 4, or 5 mismatched nucleotides at the 3' end to prevent A-tailing. Optionally or additionally, the 5' end of the stem-forming region may include one or more mismatched nucleotides that are non-hybridizable with the target-specific region. In some embodiments, the split-circular primer includes 1, 2, 3, 4, or 5 mismatched nucleotides at the 5' end of the stem-forming region.

[0076] like Figure 13 As shown, a preferred embodiment of the segmentation-circular primer according to scheme E includes, from the 5' to 3' direction, a first adaptor segment, one or more mismatched nucleotides, a stem-forming segment, a second adaptor segment, a molecular index segment, and a target-specific segment, wherein the stem-forming segment is the downstream complement of the 3' portion of the target-specific segment.

[0077] In some embodiments, the size of the stem structure formed between the 3' portion of the stem-forming region and the target-specific region is about 5-20 bp, or about 5-10 bp, or about 10-15 bp, or about 15-20 bp.

[0078] In some instances, the first connector segment is longer than the second connector segment. In some embodiments, the second connector segment is longer than the first connector segment.

[0079] In some embodiments, the segmentation-circular primers according to scheme E have preferred annealing and melting temperatures for PCR reactions, wherein the 3' portion of the stem-forming segment and the target-specific segment forms a stem structure at or below the preferred annealing temperature, but does not form a stem structure at or above the melting temperature. In some instances, preferred annealing temperatures are 60°C or below, 59°C or below, 58°C or below, 57°C or below, 56°C or below, 55°C or below, 54°C or below, 53°C or below, 52°C or below, 51°C or below, or 50°C or below. In some embodiments, melting temperatures are 60°C or above, 61°C or above, 62°C or above, 63°C or above, 64°C or above, 65°C or above, 66°C or above, 67°C or above, 68°C or above, 69°C or above, or 70°C or above. In some instances, extreme annealing temperatures, such as 30°C to 80°C, may be useful.

[0080] Primer Composition

[0081] Other embodiments of the application described herein relate to a primer composition comprising a loopable primer, a split primer, and / or a split-loopable primer described herein.

[0082] In some embodiments, the primer composition comprises at least an upstream loopable primer and a downstream loopable primer targeting the same amplification locus of interest. In some embodiments, the upstream loopable primer and the downstream loopable primer correspond to Figure 2 Scheme A, shown below. In some embodiments, the upstream loopable primer and the downstream loopable primer correspond to Figure 3 Scheme B, shown below.

[0083] In some embodiments, the primer composition comprises at least an upstream split primer and a downstream split primer targeting the same amplification locus of interest. In some embodiments, the upstream split primer and the downstream split primer correspond to Figure 3 Scheme C, shown below.

[0084] In some embodiments, the primer composition comprises at least an upstream split-loopable primer and a downstream split-loopable primer targeting the same amplification locus of interest. In some embodiments, the upstream split-loopable primer and the downstream split-loopable primer correspond to Figure 13 Scheme E, shown below. In some embodiments, the upstream split-loopable primer and the downstream split-loopable primer correspond to Figure 13 Scheme E, shown below.

[0085] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of upstream and downstream loopable primers.

[0086] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 loopable primers each comprising a different stem-forming segment. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers each comprising a different molecular index sequence. In some embodiments, the composition comprises at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different loopable primers each comprising a different combination of the stem-forming segment and the molecular index sequence.

[0087] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different partition primers. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of upstream and downstream partition primers.

[0088] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 loopable primers each comprising a different target-specific segment. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different partition primers each comprising a different molecular index sequence. In some embodiments, the composition comprises at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different partition primers each comprising a different combination of the target-specific segment and the molecular index sequence.

[0089] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split-circularizable primers. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of upstream and downstream split-circularizable primers.

[0090] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 split-circularizable primers each comprising a different stem-forming segment. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split-circularizable primers each comprising a different molecular index sequence. In some embodiments, the composition comprises at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different split-circularizable primers each comprising a different combination of the stem-forming segment and the molecular index sequence.

[0091] Nucleic acid amplification method

[0092] Other embodiments of the application described herein relate to a method for amplifying a target locus of interest from a template DNA, the method comprising performing at least two pre-amplification cycles using the circularizable primers described above or the split primers described above or the split-circularizable primers described above, wherein each pre-amplification cycle comprises annealing the primers to the template DNA or a pre-amplification product thereof and extending the annealed primers.

[0093] In some embodiments, the method comprises at least three, at least four, at least five, at least ten, or at most fifteen, or at most ten, or at most seven, or at most five pre-amplification cycles.

[0094] In some embodiments, each pre-amplification cycle comprises annealing at least an upstream circularizable primer and a downstream circularizable primer targeting the same target locus of interest to the template DNA or a pre-amplification product thereof, and extending the annealed upstream circularizable primer and the annealed downstream circularizable primer. In some embodiments, the upstream circularizable primer and the downstream circularizable primer are annealed to the same strand of the template DNA or the pre-amplification product thereof. Figure 2This corresponds to scheme A shown. In some embodiments, the upstream and downstream circulant primers are... Figure 3 This corresponds to scheme B shown.

[0095] In some embodiments, each pre-amplification cycle includes at least annealing an upstream split primer and a downstream split primer targeting the same target locus to template DNA or its pre-amplification product, and extending and annealing the upstream circularizable primer and the downstream circularizable primer. In some embodiments, the upstream circularizable primer and the downstream circularizable primer are combined with... Figure 4 This corresponds to scheme C shown.

[0096] In some embodiments, each pre-amplification cycle includes at least annealing an upstream split-circular primer and a downstream split-circular primer targeting the same target locus to template DNA or its pre-amplification product, and extending the annealed upstream split-circular primer and the annealed downstream split-circular primer. In some embodiments, the upstream split-circular primer and the downstream split-circular primer are coupled with... Figure 13 This corresponds to scheme D shown. In some embodiments, the upstream segmentation-circularizable primer and the downstream segmentation-circularizable primer are... Figure 13 This corresponds to scheme E shown.

[0097] like Figure 10 As shown, when using a pair of upstream and downstream circular primers according to scheme A, the pre-amplification product may include, for example, from the 5' to 3' direction, one or more mismatched nucleotides, a first stem-forming region, a first adaptor region, a first molecular index region, an amplified target sequence, a second molecular index region, a second adaptor region, a second stem-forming region, and one or more mismatched nucleotides.

[0098] like Figure 11 As shown, when using a pair of upstream and downstream circular primers according to scheme B, the pre-amplification product may include, for example, from the 5' to 3' direction, a first stem-forming region, one or more G / C nucleotides, a first adaptor region, a first molecular index region, an amplified target sequence, a second molecular index region, a second adaptor region, one or more G / C nucleotides, and a second stem-forming region.

[0099] like Figure 12 As shown, when using a pair of upstream and downstream split primers according to scheme C, the pre-amplified product may include, for example, from the 5' to 3' direction, a 5'-target-specific sequence, a first adaptor segment, a first molecular index segment, an amplified target sequence, a second molecular index segment, a second adaptor segment, and a 3'-target-specific sequence.

[0100] In some embodiments, the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the method further comprises a plurality of PCR cycles using one or more PCR primers that can hybridize to the universal adaptor sequence.

[0101] In some embodiments, the PCR primers comprise sequencing adaptors for high-throughput sequencing of the PCR products. In some embodiments, the PCR primers comprise sample barcodes for further analysis of the PCR product pool.

[0102] In some embodiments, the pre-amplification cycles each comprise annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers (each comprising a different stem-forming segment) to the template DNA or pre-amplification products thereof. In some embodiments, the pre-amplification cycles each comprise annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers (each comprising a different molecular index sequence) to the template DNA or pre-amplification products thereof. In some embodiments, the pre-amplification cycles each comprise annealing at least at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different loopable primers (each comprising a different combination of stem-forming segment and molecular index sequence) to the template DNA or pre-amplification products thereof.

[0103] In some embodiments, the pre-amplification cycles each comprise annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of upstream and downstream loopable primers to the template DNA or pre-amplification products thereof.

[0104] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different partition primers (each comprising a different target-specific segment) to the template DNA or pre-amplification products thereof. In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different partition primers (each comprising a different molecular index sequence) to the template DNA or pre-amplification products thereof. In some embodiments, each pre-amplification cycle comprises annealing at least at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different combinations of target-specific segments and molecular index sequences to the template DNA or pre-amplification products thereof.

[0105] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of upstream and downstream partition primers to the template DNA or pre-amplification products thereof.

[0106] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different partition- loopable primers (each comprising a different stem-forming segment) to the template DNA or pre-amplification products thereof. In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different partition- loopable primers (each comprising a different molecular index sequence) to the template DNA or pre-amplification products thereof. In some embodiments, each pre-amplification cycle comprises annealing at least at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different partition- loopable primers (each comprising a different combination of stem-forming segments and molecular index sequences) to the template DNA or pre-amplification products thereof.

[0107] In some embodiments, the pre-amplification cycles each comprise annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of upstream and downstream split-circularizable primer to the template DNA or pre-amplification products thereof.

[0108] In some embodiments, the circularizable primers according to Scheme A have a preferred annealing temperature and melting temperature for the PCR reaction, wherein the 3' portion of the stem-forming segment and the target-specific segment form a stem structure at or below the preferred annealing temperature, and do not form a stem structure at or above the melting temperature, and wherein the annealing temperature for the pre-amplification cycles is at or below the preferred annealing temperature (e.g., 60°C or below, 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below).

[0109] In some embodiments, the circularizable primers according to Scheme B have a preferred annealing temperature and melting temperature for the PCR reaction, wherein the 5' portion of the stem-forming segment and the target-specific segment form a stem structure at or below the preferred annealing temperature, and do not form a stem structure at or above the melting temperature, and wherein the annealing temperature for the pre-amplification cycles is at or below the preferred annealing temperature (e.g., 60°C or below, 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below).

[0110] In some embodiments, the split-circularizable primers according to Scheme D have a preferred annealing temperature and melting temperature for the PCR reaction, wherein the stem-forming segment and the second target-specific segment form a stem structure at or below the preferred annealing temperature, and do not form a stem structure at or above the melting temperature, and wherein the annealing temperature for the pre-amplification cycles is at or below the preferred annealing temperature (e.g., 60°C or below, 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below).

[0111] In some embodiments, the split-circularizable primers according to Scheme E have a preferred annealing temperature and a melting temperature for a PCR reaction, wherein the 5' portion of the stem-forming segment and the target-specific segment form a stem structure at or below the preferred annealing temperature, and do not form a stem structure at or above the melting temperature, and wherein the annealing temperature for the pre-amplification cycles is at or below the preferred annealing temperature (e.g., 60°C or below, or 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, or 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below).

[0112] In other embodiments, the annealing temperature for the pre-amplification cycles can be below 50°C, or below 40°C, or below 30°C, or below 20°C, or above 60°C, or above 65°C, or above 70°C. In other embodiments, extreme annealing temperatures can be useful for the pre-amplification cycles, such as 30°C to 80°C.

[0113] Nucleic acid amplification kit

[0114] Other embodiments of the application described herein relate to a kit for amplifying a target locus of interest from a template DNA, comprising the above-described circularizable primers or the above-described split primers or the above-described split-circularizable primers.

[0115] In some embodiments, the kit comprises at least an upstream circularizable primer and a downstream circularizable primer that target the same locus of interest for amplification. In some embodiments, the kit comprises at least an upstream split primer and a downstream split primer that target the same locus of interest for amplification. In some embodiments, the kit comprises at least an upstream split-circularizable primer and a downstream split-circularizable primer that target the same locus of interest for amplification.

[0116] In some embodiments, the kit further comprises a polymerase for extending the circularizable primers or the split primers or the split-circularizable primers in the pre-amplification cycles.

[0117] In some embodiments, the kit further comprises a protease for inactivating the aforementioned polymerase after completion of the pre-amplification cycles.

[0118] In some embodiments, the kit further comprises one or more PCR primers that can hybridize to a universal adapter in the adapter segment of the circularizable primers or the split primers or the split-circularizable primers. In some embodiments, the PCR primers comprise sequencing adapters for high-throughput sequencing of the PCR products. In some embodiments, the PCR primers comprise sample barcodes for further analysis of the PCR product pool.

[0119] Applications

[0120] The loopable primers of Scheme A (3'-target-stem loop) can hide / protect the universal adaptor sequence and MIT sequences in highly multiplexed PCR improve assay specificity by inhibiting primer dimer and non-specific binding. Thus, the loopable primers of Scheme A are particularly useful for the following applications:

[0121] Copy number variation detection (CNV, aneuploidy, microdeletion, etc.): Each DNA fragment product carries a unique (or unique combination) molecular indexing tag, so that the number of fragments in a particular locus (amplicon sequence) sample can be traced.

[0122] PCR error removal, true mutation detection: By using MIT barcodes, PCR products such as sequence changes produced by polymerase errors that do not exist in the original molecule can be identified and separated from true variants / mutations that exist in the original molecule.

[0123] PCR tiling: The stem at the 3' end of the primer prevents the formation of primer dimers, which is very useful for amplifying overlapping or tiled amplicons in a single multiplexed PCR reaction.

[0124] Allele-specific amplification: Mutant base positions in the allele-specific primer located in the stem region (3'-end of primer) will inhibit mismatched wild type to open the stem, thereby preventing wild type amplification.

[0125] Mutant allele-specific quantitative PCR (qPCR) and digital PCR (qPCR): Mutant and wild type primers have different tag sequences, which can be detected by different fluorescent probe colors and other detection methods.

[0126] Another embodiment of the application described herein relates to a method of determining a variant copy number of a target locus of interest, the method comprising: pre-amplifying the target locus of interest from a template DNA using at least two pre-amplification cycles with one or more loopable primers, each of the loopable primers comprising a target-specific segment, an adaptor segment, a molecular indexing segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the molecular indexing segment comprises a molecular indexing sequence; amplifying the pre-amplification product using one or more PCR primers hybridizable to the universal adaptor sequence; and sequencing the amplification product to determine the variant copy number of the target locus of interest using the molecular indexing sequence.

[0127] Another embodiment of the invention described herein relates to a method for determining fetal aneuploidy, the method comprising: pre-amplifying a plurality of target loci of interest of one or more chromosomes from cell-free DNA isolated from a maternal blood sample using at least two pre-amplification cycles employing a plurality of loopable primers, each comprising a target-specific segment, an adaptor segment, a molecular index segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the molecular index segment comprises a molecular index sequence; amplifying the pre-amplification products using one or more PCR primers hybridizable to the universal adaptor sequence; and sequencing the amplification products to determine fetal aneuploidy using the molecular index sequence.

[0128] Another embodiment of the invention described herein relates to a method of multiplex amplification, the method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles employing at least a first loopable primer and a second loopable primer, each comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the first and second loopable primers comprise complementary sequences of their target-specific segments and are capable of forming a primer dimer lacking protection by the stem-forming segment; and amplifying the pre-amplification products using one or more PCR primers hybridizable to the universal adaptor sequence.

[0129] Another embodiment of the invention described herein relates to a method of allele-specific amplification, the method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles employing a loopable primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure, wherein the adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the loopable primer comprises a SNV or SNP allele at the 5' portion or the 3' portion of the target-specific segment; and amplifying the pre-amplification products using one or more PCR primers hybridizable to the universal adaptor sequence.

[0130] Another embodiment of the application described herein relates to an allele-specific quantitative PCR (qPCR) method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles employing at least a first and a second loopable primer, each comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion, and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure, wherein the adaptor segment of the first loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adaptor segment of the second loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' portion or the 3' portion of the target-specific segment of the first loopable primer comprises a first SNV or SNP allele, and wherein the 5' portion or the 3' portion of the target-specific segment of the second loopable primer comprises a second SNV or SNP allele; amplifying the pre-amplification product using one or more PCR primers hybridizable to the universal adaptor sequence in the presence of the first and the second fluorescent probes; and detecting real-time fluorescent signal intensity from the first and the second fluorescent probes. Alternatively, the method for allele-specific qPCR does not require a pre-amplification step, but comprises amplifying one or more target loci of interest from template DNA using the first and the second loopable primers in the presence of the first and the second fluorescent probes; and detecting real-time fluorescent signal intensity from the first and the second fluorescent probes.

[0131] Another embodiment of the application described herein relates to an allele-specific digital PCR (dPCR) method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles employing at least a first loopable primer and a second loopable primer, each comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein the target-specific segment comprises a 5' portion and a 3' portion and the stem-forming segment is hybridizable to the 3' portion of the target-specific segment to form a stem structure, wherein the adaptor segment of the first loopable primer comprises a universal adaptor sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adaptor segment of the second loopable primer comprises a universal adaptor sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' portion or the 3' portion of the target-specific segment of the first loopable primer comprises a first SNV or SNP allele, and wherein the 5' portion or the 3' portion of the target-specific segment of the second loopable primer comprises a second SNV or SNP allele; partitioning the pre-amplification product into a plurality of reaction volumes; amplifying the pre-amplification product in each reaction volume using one or more PCR primers hybridizable to the universal adaptor sequence in the presence of the first fluorescent probe and the second fluorescent probe; and detecting the presence or absence of fluorescent signals from the first fluorescent probe and the second fluorescent probe. Alternatively, the method for allele-specific dPCR does not require a pre-amplification step, but comprises partitioning a sample into a plurality of reaction volumes; amplifying one or more target loci of interest from template DNA in each reaction volume using a first and a second loopable primer in the presence of a first and a second fluorescent probe; and detecting the presence or absence of fluorescent signals from the first and the second fluorescent probe.

[0132] Working Example

[0133] Example 1: 2-cycle workflow

[0134] As shown in Figure 5 , a proof-of-concept experiment was conducted to amplify sample DNA using a 2-cycle workflow. For each combination of primer schemes, Figure 1 , commercially available high-fidelity enzyme mix and DNA were prepared.

[0135] Premix Initial vol [μl] Final 1.6X enzyme mix 1.6 X 6.25 1 X 4X primer mix 4 X 2.5 1 X DNA 1.25 1 X Final 10

[0136] * All tested polymerase formulations were identical.

[0137] MIT by direct PCR reaction: samples were cycled under the following conditions. After 2 cycles, 20 μΐ^of prepared protease solution was added to the reaction and incubated at 65 °C for 15 minutes, then mismatch inactivated at 95 °C for 15 minutes.

[0138]

[0139]

[0140] Sequencing barcoding reaction: 10 μΐ^of the resulting volume was placed in a Q5 barcoding reaction and cycled 35 times to full plateau.

[0141]

[0142]

[0143] Step Cycle number Temperature Time Hold 1 98℃ 3 min Cycle 35 98℃ 30 sec 62.5℃ 30 sec MAX mode 72℃ 30 sec Hold 1 72℃ 2 min Hold 1 4℃ Infinite

[0144] Pooling and purification: 2 μΐ^of each sample was pooled together and purified using Qiagen Qiaquick spin columns in 50 μΐ^pools.

[0145]

[0146] Samples were quantified by qPCR and sequenced.

[0147] As shown in Figure 6 , each of Protocol A, Protocol B, and Protocol C was able to amplify the target locus of interest with 2 pre-amplification cycles (followed by downstream PCR amplification using primers that can hybridize to universal adaptor sequences), with Protocol A showing the best on-target rate. As shown in Figure 9 , the MIT counts were very consistent between replicate samples (Protocol A, 2 pre-amplification cycles).

[0148] Example 2: 3 / 10 cycle workflow

[0149] As shown in Figure 7 , proof of concept experiments were carried out to amplify sample DNA using either a 3 cycle or 10 cycle workflow. For each combination of primer protocols (see Figure 1 ), commercially available high fidelity enzyme mixes and DNA were prepared.

[0150] Premix Initial vol [μl] Final 1.6X enzyme mix 1.6 X 6.25 1 X 4X primer mix 4 X 2.5 1 X DNA 1.25 1 X Final 10

[0151] * All tested polymerase formulations were identical.

[0152] MIT by direct PCR reaction: samples were cycled under the following conditions. After 3 or 10 cycles, 20 μΐ, of prepared protease solution was added to the reaction and incubated at 65 °C for 15 minutes, then inactivated at 95 °C for 15 minutes.

[0153]

[0154]

[0155] Sequencing barcoding reaction: 10 μΐ, of the resulting volume was placed in a Q5 barcoding reaction and cycled 35 times to full plateau.

[0156]

[0157] Step Cycle number Temperature Time Hold 1 98℃ 3 min Cycle 35 98℃ 30 sec 62.5 30 sec MAX mode 72℃ 30 sec Hold 1 72℃ 2 min Hold 1 4℃ Infinite

[0158] Pooling and purification: 2 μΐ, of each sample was pooled together and purified using Qiagen Qiaquick spin columns in 50 μΐ, pools.

[0159]

[0160]

[0161] Samples were quantified by qPCR and sequenced.

[0162] As shown in Figure 8 schemes A, B, and C each enable amplification of a target locus of interest by 3 or 10 pre-amplification cycles (followed by downstream PCR amplification using primers that can hybridize to universal adaptor sequences), scheme A shows the best on-target rate at 3 pre-amplification cycles, and scheme C shows the best on-target rate at 10 pre-amplification cycles.

[0163] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the application disclosed herein without departing from the scope and spirit of the application. The application illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that is not specifically disclosed herein. The use of any and all examples, or exemplary language (e.g., "such as", "for example", "e.g.", "for instance", "as follows", and the like), is merely intended to better illuminate the application and does not pose a limitation on the scope of the application unless specifically claimed. It should be understood that the application is not limited in scope or spirit to the particular arrangements described, and that various modifications can be made without departing from the scope and nature of the application as described. SEQUENCE LISTING <110> NATERA, INC. <120> New primers and uses thereof <130> N.022.WO.01 <140> <141> <150> 62 / 617,066 <151> 2018-01-12 <160> 9 <170> PatentIn 3.5 version <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "Artificial sequence description: synthetic primer" <220> <221> Modified Bases <222> (21)..(24) <223> a, c, t, g, unknown, or other <400> 1 acacgacgct cttccgatct nnnn 24 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "Artificial sequence description: synthetic primer" <220> <221> Modified Bases <222> (22)..(25) <223> a, c, t, g, unknown, or other <400> 2 agacgtgtgc tcttccgatc tnnnn 25 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic sequence description: synthetic oligonucleotide” <220> <221> modified base <222> (1)..(2) <223> a, c, t, g, unknown, or other <220> <221> modified base <222> (23)..(29) <223> a, c, t, g, unknown, or other <400> 3 nnacacgacg ctcttccgat ctnnnnnnn 29 <210> 4 <211> 30 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic sequence description: synthetic oligonucleotide” <220> <221> modified base <222> (1)..(7) <223> a, c, t, g, unknown, or other <220> <221> modified base <222> (29)..(30) <223> a, c, t, g, unknown, or other <400> 4 nnnnnnnaga tcggaagagc acacgtctnn 30 <210> 5 <211> 28 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic sequence description: synthetic primer” <220> <221> modified base <222> (23)..(26) <223> a, c, t, g, unknown, or other <400> 5 ggacacgacg ctcttccgat ctnnnncc 28 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <221> Origin <223> NOTE="Artificial sequence description: synthetic primer" <220> <221> Modified Bases <222> (24)..(27) <223> a, c, t, g, unknown, or other <400> 6 ggagacgtgt gctcttccga tctnnnncc 29 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <221> Origin <223> NOTE="Artificial sequence description: synthetic oligonucleotide" <220> <221> Modified Bases <222> (1)..(2) <223> a, c, t, g, unknown, or other <220> <221> Modified Bases <222> (25)..(28) <223> a, c, t, g, unknown, or other <220> <221> Modified Bases <222> (31)..(33) <223> a, c, t, g, unknown, or other <400> 7 nnggacacga cgctcttccg atctnnnncc nnn 33 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <221> Source <223> NOTE="Artificial Sequence description: Synthetic oligonucleotide" <220> <221> Modified Base <222> (1)..(3) <223> a, c, t, g, unknown, or other <220> <221> Modified Base <222> (6)..(9) <223> a, c, t, g, unknown, or other <220> <221> Modified Base <222> (33)..(34) <223> a, c, t, g, unknown, or other <400> 8 nnnggnnnn agatcggaag agcacacgtc tccnn 34 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> Source <223> NOTE="Artificial Sequence description: Synthetic oligonucleotide" <220> <221> Modified Base <222> (1)..(2) <223> a, c, t, g, unknown, or other <220> <221> Modified Base <222> (23)..(28) <223> a, c, t, g, unknown, or other <400> 9 nnacacgacg ctcttccgat ctnnnnnn 28

Claims

1. A composition comprising a primer, the primer being: A loopable primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein, the stem-forming segment is hybridizable to a portion of the target-specific segment to form a stem structure, wherein the target-specific segment comprises a 5' portion and a 3' portion, the hybridization between the stem-forming segment and the portion of the target-specific segment forms a loop, the loop comprising the adapter segment and the 5' portion of the target-specific segment, and wherein the loopable primer comprises one or more mismatched nucleotides at the 3' end of the target-specific segment that are not hybridizable to one or more mismatched nucleotides at the 5' end of the stem-forming segment.

2. The composition of claim 1, wherein, the primer further comprises a molecular index segment, the molecular index segment comprising a molecular index sequence.

3. The composition of claim 2, wherein, the molecular index segment is located between the target-specific segment and the adapter segment.

4. The composition of claim 3, wherein, the molecular index segment is located 5' to the target-specific segment and 3' to the adapter segment, and wherein the hybridization between the stem-forming segment and the portion of the target-specific segment forms a loop, the loop comprising the adapter segment and the molecular index segment.

5. The composition of claim 1, wherein, the loopable primer further comprises one or more of a G or C nucleotide located 5' to the target-specific segment for stabilizing the stem structure with one or more complementary G or C nucleotides.

6. The composition of claim 1, comprising at least an upstream loopable primer and a downstream loopable primer that target the same amplification locus of interest.

7. The composition of claim 1, wherein, the adapter segment comprises a universal adapter sequence for PCR amplification and / or sequencing.

8. The composition of claim 1, comprising at least 50 different primers, each comprising a different stem-forming segment.

9. The composition of claim 1, comprising at least 50 different primers, each comprising a different molecular index sequence.

10. The composition of claim 1, comprising at least 2,500 different primers, each comprising a different combination of the stem-forming segment and molecular index sequence.

11. A method of amplifying a target locus of interest from a template DNA, the method comprising at least two pre-amplification cycles using a primer, the primer being: A loopable primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein, the stem-forming segment is hybridizable to a portion of the target-specific segment to form a stem structure, wherein the target-specific segment comprises a 5' portion and a 3' portion, the hybridization between the stem-forming segment and the portion of the target-specific segment forms a loop, the loop comprising the adapter segment and the 5' portion of the target-specific segment, and wherein the loopable primer comprises one or more mismatched nucleotides at the 3' end of the target-specific segment that are not hybridizable to one or more mismatched nucleotides at the 5' end of the stem-forming segment; wherein each pre-amplification cycle comprises annealing the primer to the template DNA or a pre-amplification product thereof and extending the annealed primer.

12. The method of claim 11, wherein, the method comprises three or more pre-amplification cycles using the loopable primer.

13. The method of claim 11, wherein, The method includes five or more pre-amplification cycles using the loopable primers.

14. The method of claim 11, wherein, The method includes ten or fewer pre-amplification cycles using the loopable primers.

15. The method of claim 11, wherein, The primers further include a molecular index segment comprising a molecular index sequence.

16. The method of claim 15, wherein, The molecular index segment is located between the target-specific segment and the adaptor segment.

17. The method of claim 16, wherein, The molecular index segment is located 5' to the target-specific segment and 3' to the adaptor segment, and wherein hybridization between the stem-forming segment and a portion of the target-specific segment forms a loop comprising the adaptor segment and the molecular index sequence.

18. The method of claim 11, wherein, The loopable primers further include one or more of a G or C nucleotide 5' to the target-specific segment for stabilizing the stem structure with one or more complementary G or C nucleotides.

19. The method of claim 11, wherein, Each of the pre-amplification cycles includes annealing at least an upstream loopable primer and a downstream loopable primer targeting the same locus of interest to the template DNA or a pre-amplification product thereof, and extending the annealed upstream loopable primer and the annealed downstream loopable primer.

20. The method of claim 11, wherein, The adaptor segment comprises a universal adaptor sequence for PCR amplification, and wherein the method further includes a plurality of PCR cycles using one or more PCR primers hybridizable to the universal adaptor sequence.

21. The method of claim 20, wherein, Each of the one or more PCR primers comprises a sequencing adaptor and / or a sample barcode.

22. The method of claim 11, wherein, Each of the pre-amplification cycles includes annealing at least 50 different primers to the template DNA or a pre-amplification product thereof, the different primers each comprising a different stem-forming segment.

23. The method of claim 11, wherein, Each of the pre-amplification cycles includes annealing at least 50 different primers to the template DNA or a pre-amplification product thereof, the different primers each comprising a different molecular index sequence.

24. The method of claim 11, wherein, Each of the pre-amplification cycles includes annealing at least 2,500 different primers to the template DNA or a pre-amplification product thereof, the different primers each comprising a different combination of the stem-forming segment and a molecular index sequence.

25. A kit for amplifying a target locus of interest, comprising primers, the primers being: A loopable primer comprising a target-specific segment, an adaptor segment, and a stem-forming segment, wherein, The stem-forming segment is hybridizable to a portion of the target-specific segment to form a stem structure, wherein the target-specific segment comprises a 5' portion and a 3' portion, the hybridization between the stem-forming segment and the portion of the target-specific segment forms a loop comprising the adaptor segment and the 5' portion of the target-specific segment, and wherein the loopable primer comprises one or more mismatched nucleotides at the 3' end of the target-specific segment, the mismatched nucleotides being non-hybridizable to one or more mismatched nucleotides at the 5' end of the stem-forming segment.

26. The kit of claim 25, further comprising a polymerase.

27. The kit of claim 25, further comprising a protease.

28. The kit of claim 25, further comprising one or more PCR primers, the PCR primers being hybridizable to a universal adaptor sequence.

29. The kit of claim 28, wherein, The one or more PCR primers each include a sequencing adapter and / or a sample barcode.

30. The kit of claim 25, wherein, The primers further include a molecular index segment that includes a molecular index sequence.

31. The kit of claim 30, wherein, The molecular index segment is located between the target-specific segment and the adapter segment.

32. The kit of claim 31, wherein, The molecular index segment is located 5' to the target-specific segment and 3' to the adapter segment, and wherein hybridization between the stem-forming segment and a portion of the target-specific segment forms a loop that includes the adapter segment and the molecular index sequence.

33. The kit of claim 25, wherein, The loopable primer further includes one or more of a G or C nucleotide located 5' to the target-specific segment for stabilizing the stem structure with one or more complementary G or C nucleotides.

34. The kit of claim 25, comprising at least an upstream loopable primer and a downstream loopable primer that target the same amplification locus of interest.

35. The kit of claim 25, wherein, The adapter segment includes a universal adapter sequence for PCR amplification and / or sequencing.

36. The kit of claim 25, comprising a pool of at least 50 different primers each including a different stem-forming segment.

37. The kit of claim 25, comprising a pool of at least 50 different primers each including a different molecular index sequence.

38. The kit of claim 25, comprising a pool of at least 2,500 different primers each including a different combination of the stem-forming segment and molecular index sequence.

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