Control of Tagmentation Library Insert Sizes Using Archaeal Histone-Like Proteins

Abstract

The present disclosure provides compositions and kits for the tagmentation of double-stranded DNA. In some embodiments, the compositions and kits for the tagmentation of double-stranded DNA include one or more histone-like proteins and / or one or more transposition systems. The present disclosure also provides methods for the tagmentation of double-stranded DNA in the presence of one or more histone-like proteins.

Description

Background Art

[0001] Background of the Disclosure The advent of single-cell genome amplification technology and next-generation sequencing methods has provided a breakthrough in the ability to sequence the genomes and transcriptomes of individual biological cells. Large-scale parallel DNA sequencing of thousands of samples in a single instrument run is now possible, but the preparation of individual sequencing libraries is expensive and time-consuming. Library preparation is an essential process that precedes the sequencing itself and involves several aspects that affect the efficiency of sequencing. Library preparation "usually includes the following main steps: fragmentation of the input DNA, end repair and A-tailing of the DNA fragments, ligation of indexed sequencing adapters, and optional amplification of the ligation products." (Ribarska et al., 'Optimization of enzymatic fragmentation is crucial to maximize genome coverage: a comparison of library preparation methods for Illumina sequencing,' BMC Genomics. 2022;23:92).

[0002] One of the major obstacles to sample preparation is DNA fragmentation. The size of the DNA fragments generated depends on the sequencing platform used, ranging from a few hundred base pairs for short-read sequencing technologies (e.g., Illumina®, Ion Torrent™) to fragments over 10 kb for long-read sequencing technologies (e.g., Pacific Biosciences® and Oxford Nanopore Technologies®). Methods for fragmenting DNA can be broadly classified into two basic categories: mechanical and enzyme-based. Mechanical shearing methods include acoustic shearing, hydrodynamic shearing, and nebulization, while enzyme-based methods include transposons, restriction enzymes, and nicking enzymes. Although there are many different options for fragmenting DNA, when choosing a fragmentation method, the final fragment size, the amount of starting material, upfront equipment investment, and scalability must be considered. Importantly, for use in next-generation sequencing, the method employed must shear the DNA randomly enough so that the sequenced library fully represents the original sample.

[0003] Tagmentation is a process that combines fragmentation and adapter incorporation steps. As used herein, the term "tagmenting" refers to transposase-catalyzed fragmentation of a double-stranded DNA sample and tagging of the fragments with sequences adjacent to the transposon end sequences. A hyperactive mutant of the bacterial Tn5 transposase that mediates fragmentation of double-stranded DNA and ligates synthetic oligonucleotides is widely used in next-generation sequencing (NGS). Its usefulness in generating libraries for NGS systems was first described in a paper by Andrew Adey et al. in 2010 (the disclosure of which is incorporated herein by reference in its entirety: Adey et al., "Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition," Genome Biol 11:R119, 2010). In commercial products such as Illumina's Nextera and Thermo Scientific's MuSeek, the transposase inserts NGS system-specific adapter oligonucleotides into the double-stranded DNA sample. Such a simple one-step tagmentation reaction has significantly simplified the process of preparing libraries for sequencing, shortened the workflow time, and reduced costs. Summary of the Invention

[0004] Brief Summary of the Disclosure The approach for array determination using tagmentation involves fragmentation of double-stranded DNA while adding universal overhangs. As described above, the workflow enables rapid generation of sequencing libraries for this combined fragmentation and tagging process. However, the sequencing library preparation process is highly sensitive to the input DNA concentration and / or the concentration of the input transposition system. Therefore, accurate quantification of the input DNA concentration and / or the concentration of the transposition system is required to control the fragment size. The applicant has unexpectedly discovered that tagmentation in the presence of one or more histone-like proteins reduces the need for accurate quantification of the input DNA concentration and / or the concentration of the transposition system. Furthermore, the applicant has discovered that tagmentation in the presence of one or more histone-like proteins enables precise control of the DNA fragment size distribution. Additionally, the applicant has discovered that tagmentation in the presence of one or more histone-like proteins allows for better control of the tagmentation fragment size, facilitating greater utility of tagmentation in applications that require longer fragment inserts.

[0005] A first aspect of the present disclosure is a composition comprising a histone-like protein and a transposition system. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaea. In some embodiments, the histone-like protein is an archaeal histone-like protein derived from either the genus Thermococcus or the genus Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 7.5 ng / μL to about 20 ng / μL.

[0006] In some embodiments, the transposition system comprises a transposase and an adapter. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase and a Tn5 transposase recognition site. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase, a Tn5 transposase recognition site, and one or more oligonucleotides. In some embodiments, the concentration of the transposition system in the composition ranges from about 150 ng / μL to about 200 ng / μL. In some embodiments, the concentration of the transposition system in the composition ranges from about 180 ng / μL.

[0007] In some embodiments, the composition further comprises nucleic acid sequences such as DNA, ctDNA, double-stranded DNA. In some embodiments, the double-stranded DNA is derived from a human subject. In some embodiments, the double-stranded DNA is derived from a tumor sample. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is about 5 ng / μL. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 0.5:1 to about 5:1. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 1:1 to about 4:1. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 1.5:1 to about 3:1.

[0008] In some embodiments, the composition further comprises a divalent cation. In some embodiments, the divalent cation is selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+, and Ca2+. In some embodiments, the divalent cation is Mn2+. In some embodiments, the composition further comprises an LMW buffer. In some embodiments, the LMW buffer comprises Tris-acetate, glycerol, and DMSO.

[0009] A second aspect of the present disclosure is a composition comprising a histone-like protein, a transposition system, and double-stranded DNA. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaea. In some embodiments, the histone-like protein is an archaeal histone-like protein derived from either the genus Thermococcus or the genus Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 7.5 ng / μL to about 20 ng / μL.

[0010] In some embodiments, the transposition system comprises a transposase, a transposon, and an adapter. In some embodiments, the transposition system comprises TnAa. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase and a Tn5 transposase recognition site. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase, a Tn5 transposase recognition site, and one or more oligonucleotides. In some embodiments, the concentration of the transposition system in the composition ranges from about 150 ng / μL to about 200 ng / μL. In some embodiments, the concentration of the transposition system in the composition ranges from about 180 ng / μL.

[0011] In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is about 5 ng / μL. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 0.5:1 to about 5:1. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 1:1 to about 4:1. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 1.5:1 to about 3:1.

[0012] In some embodiments, the composition further comprises a divalent cation. In some embodiments, the divalent cation is selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+, and Ca2+. In some embodiments, the divalent cation is Mn2+. In some embodiments, the composition further comprises an LMW buffer. In some embodiments, the LMW buffer comprises Tris-acetate, glycerol, and DMSO.

[0013] A third aspect of the present disclosure is a composition comprising a histone-like protein, a transposition system, double-stranded DNA, and a divalent cation (e.g., Co2+, Mn2+, Mg2+, Cd2+, and Ca2+). In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaeon. In some embodiments, the histone-like protein is an archaeal histone-like protein derived from either the genus Thermococcus or the genus Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of the histone-like protein in the composition ranges from about 7.5 ng / μL to about 20 ng / μL.

[0014] In some embodiments, the transposition system comprises a transposase, a transposon, and an adapter. In some embodiments, the transposition system comprises TnAa. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase and a Tn5 transposase recognition site. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase, a Tn5 transposase recognition site, and one or more oligonucleotides. In some embodiments, the concentration of the transposition system in the composition ranges from about 150 ng / μL to about 200 ng / μL. In some embodiments, the concentration of the transposition system in the composition ranges from about 180 ng / μL.

[0015] In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is about 5 ng / μL. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 0.5:1 to about 5:1. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 1:1 to about 4:1. In some embodiments, the ratio of the concentration of histone-like protein to the concentration of DNA in the composition ranges from about 1.5:1 to about 3:1.

[0016] A fourth aspect of the present disclosure is a kit comprising a first container containing a histone-like protein and a second container containing a transposition system. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaea. In some embodiments, the histone-like protein is an archaeal histone-like protein derived from either the genus Thermococcus or the genus Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2. In some embodiments, the concentration of histone-like protein in the composition ranges from about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of histone-like protein in the first container ranges from about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of histone-like protein in the first container ranges from about 7.5 ng / μL to about 20 ng / μL.

[0017] In some embodiments, the transposition system includes a transposase and an adapter. In some embodiments, the transposition system includes a hyperactive Tn5 transposase and a Tn5 transposase recognition site. In some embodiments, the transposition system includes a hyperactive Tn5 transposase, a Tn5 transposase recognition site, and one or more oligonucleotides. In some embodiments, the concentration of the transposition system in the second container ranges from about 150 ng / μL to about 200 ng / μL. In some embodiments, the transposition system concentration in the second container ranges from about 180 ng / μL. In some aspects, the kit further includes a third container containing a divalent cation and / or one or more PCR reagents (e.g., polymerase).

[0018] A fifth aspect of the present disclosure is a method for tagging double-stranded DNA, comprising: (i) obtaining a sample containing double-stranded DNA; (ii) introducing a fragmentation composition containing a histone-like protein and a transposition system into the obtained sample to provide a fragmentation reaction mixture; (iii) heating the fragmentation reaction mixture at a predetermined temperature for a predetermined time; and (iv) isolating the tagged DNA from the fragmentation reaction mixture. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaeon. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2. In some embodiments, the concentration of the histone-like protein in the fragmentation reaction mixture ranges from about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of the histone-like protein in the fragmentation reaction mixture ranges from about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of the histone-like protein in the fragmentation reaction mixture ranges from about 7.5 ng / μL to about 20 ng / μL.

[0019] In some embodiments, the transposition system includes a transposase, a transposon, and an adapter. In some embodiments, the transposition system includes a hyperactive Tn5 transposase and a Tn5 transposase recognition site. In some embodiments, the transposition system includes a hyperactive Tn5 transposase, a Tn5 transposase recognition site, and one or more oligonucleotides. In some embodiments, the concentration of the transposase in the fragmentation reaction mixture ranges from about 150 ng / μL to about 200 ng / μL. In some embodiments, the concentration of the transposase in the fragmentation reaction mixture is about 180 ng / μL.

[0020] In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is about 5 ng / μL. In some embodiments, the ratio of the concentration of the histone-like protein to the concentration of double-stranded DNA in the fragmentation reaction mixture ranges from about 0.5:1 to about 5:1. In some embodiments, the ratio of the concentration of the histone-like protein to the concentration of double-stranded DNA in the fragmentation reaction mixture ranges from about 1:1 to about 4:1. In some embodiments, the ratio of the concentration of the histone-like protein to the concentration of double-stranded DNA in the fragmentation reaction mixture ranges from about 1.5:1 to about 3:1.

[0021] In some embodiments, the predetermined time ranges from about 2 minutes to about 10 minutes. In some embodiments, the predetermined time ranges from about 3 minutes to about 7 minutes. In some embodiments, the predetermined time is about 5 minutes. In some embodiments, the predetermined temperature is from about 40°C to about 60°C. In some embodiments, the predetermined temperature is from about 45°C to about 55°C. In some embodiments, the predetermined temperature is from about 50°C to about 55°C.

[0022] In some embodiments, the method further includes introducing a stop solution into the reaction mixture. In some embodiments, the stop solution contains SDS.

[0023] In some embodiments, the isolation of the tagged DNA comprises: (a) capturing the generated DNA fragments onto beads; (b) flushing impurities from the reaction mixture; and (c) eluting the captured DNA fragments from the beads. In some embodiments, the isolated tagged DNA has a size distribution in the range of about 250 bp to about 300 bp. In some embodiments, the method further includes amplifying the isolated double-stranded DNA fragments to provide a plurality of amplicons. In some aspects, the method further includes sequencing the plurality of amplicons.

[0024] A sixth aspect of the present disclosure is a method for processing a sample containing genomic material, the method comprising: (i) obtaining a tagmentation reaction mixture comprising a transposition system and optionally a buffer; (ii) introducing into the tagmentation reaction mixture a solution comprising one or more nucleosome-like structures to provide a fragmentation reaction mixture, wherein the one or more nucleosome-like structures comprise double-stranded DNA entwined or wrapped around one or more histone-like proteins, the step of introducing a solution comprising one or more nucleosome-like structures to provide a fragmentation reaction mixture; and (iii) heating the fragmentation reaction mixture at a predetermined temperature for a predetermined time. In some embodiments, the double-stranded DNA and the histone-like protein are first mixed together to form a DNA-histone-like protein solution, and then the DNA-histone-like protein solution is added to the tagmentation reaction mixture to provide a fragmentation reaction mixture. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaeon. In some embodiments, the histone-like protein is an archaeal histone-like protein derived from either the genus Thermococcus or the genus Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2.

[0025] In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is about 5 ng / μL. In some embodiments, the ratio of the concentration of the histone-like protein in the tagmentation reaction mixture to the concentration of double-stranded DNA in the fragmentation reaction mixture ranges from about 1:1 to about 4:1. In some embodiments, the ratio ranges from about 1.5:1 to about 3:1.

[0026] A seventh aspect of the present disclosure is a method for processing a sample containing genomic material, comprising: (i) obtaining a sample in a reaction vessel, wherein the sample contains double-stranded DNA material; (ii) introducing a histone-like protein into the reaction vessel to provide a DNA histone-like protein solution; (iii) introducing a transposition system into the DNA histone-like protein solution to provide a fragmentation reaction mixture; and (iv) heating the fragmentation reaction mixture of the sample to a predetermined temperature for a predetermined time. In some embodiments, the transposition system comprises a hyperactive mutant of TnAa or Tn5 transposase and an oligonucleotide material. In some embodiments, the oligonucleotide material comprises synthetic oligonucleotides. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaeon. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2.

[0027] In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is about 5 ng / μL.

[0028] In some embodiments, the concentration of histone-like protein in the fragmentation reaction mixture ranges from about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of histone-like protein in the fragmentation reaction mixture ranges from about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of histone-like protein in the fragmentation reaction mixture ranges from about 7.5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of histone-like protein in the fragmentation reaction mixture ranges from about 10 ng / μL to about 20 ng / μL. In some embodiments, the concentration of histone-like protein in the fragmentation reaction mixture ranges from about 10 ng / μL to about 15 ng / μL. In some embodiments, the concentration of histone-like protein in the fragmentation reaction mixture ranges from about 15 ng / μL to about 20 ng / μL.

[0029] In some embodiments, the predetermined time ranges from about 2 minutes to about 10 minutes. In some embodiments, the predetermined time ranges from about 3 minutes to about 7 minutes. In some embodiments, the predetermined time is about 5 minutes. In some embodiments, the predetermined temperature is from about 40°C to about 60°C. In some embodiments, the predetermined temperature is from about 45°C to about 55°C. In some embodiments, the predetermined temperature is from about 50°C to about 55°C.

[0030] In some embodiments, the method further includes isolating the tagged DNA from the fragmentation reaction mixture. In some embodiments, the isolated tagged DNA has a size distribution in the range of about 250 bp to about 350 bp. In some embodiments, the isolated tagged DNA has a size distribution in the range of about 250 bp to about 320 bp. In some embodiments, the isolated tagged DNA has a size distribution in the range of about 250 bp to about 300 bp. In some embodiments, the method further includes amplifying the isolated tagged DNA to provide a plurality of amplicons. In some embodiments, the method further includes sequencing the plurality of amplicons. In some embodiments, the sequencing of the amplicons includes next-generation sequencing.

[0031] The eighth aspect of the present disclosure is a double-stranded DNA fragment having a size in the range of about 250 to about 350 bp, wherein the double-stranded DNA fragment is prepared by a method comprising: (i) obtaining a sample containing double-stranded DNA; (ii) introducing a fragmentation composition comprising a histone-like protein and a transposition system into the obtained sample to provide a fragmentation reaction mixture; and (iii) heating the fragmentation reaction mixture at a predetermined temperature for a predetermined time. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaeon. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2.

[0032] In some embodiments, the concentration of the histone-like protein in the fragmentation reaction mixture ranges from about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of the histone-like protein in the fragmentation reaction mixture ranges from about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of the histone-like protein in the reaction mixture ranges from about 7.5 ng / μL to about 20 ng / μL. In some embodiments, the transposition system includes a transposase, a transposon, and an adapter. In some embodiments, the concentration of the transposition system in the fragmentation reaction mixture ranges from about 150 ng / μL to about 200 ng / μL.

[0033] In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is about 5 ng / μL. In some embodiments, the ratio of the concentration of the histone-like protein to the concentration of double-stranded DNA in the fragmentation reaction mixture ranges from about 0.5:1 to about 5:1. In some embodiments, the ratio of the concentration of the histone-like protein to the concentration of double-stranded DNA in the fragmentation reaction mixture ranges from about 1:1 to about 4:1.

[0034] In some embodiments, the preparation further includes removing impurities from the reaction mixture. In some embodiments, the removal of impurities includes: (a) capturing DNA fragments on beads; (b) flushing impurities from the reaction mixture; and (c) eluting the captured DNA fragments from the beads.

[0035] A ninth aspect of the present disclosure is a fragmentation composition comprising one or more histone-like proteins, one or more transposition systems, and at least one additional component. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2. In some embodiments, the at least one additional component is selected from a buffer, a polyol, a salt, or DMSO.

[0036] A tenth aspect of the present disclosure is a kit comprising a fragmentation composition and a double-stranded DNA sample.

[0037] An eleventh aspect of the present disclosure is a kit comprising a fragmentation composition and a polymerase.

[0038] A twelfth aspect of the present disclosure is a kit comprising a fragmentation composition and a next-generation sequencing device.

[0039] A thirteenth aspect of the present disclosure is a kit comprising a fragmentation composition and one or more reagents for performing a polymerase chain reaction.

[0040] A fourteenth aspect of the present disclosure is a kit comprising a fragmentation composition that is a solution containing one or more divalent cations.

[0041] A fifteenth aspect of the present disclosure is a composition comprising a histone-like protein, a transposase, a transposon end composition, and one or more oligonucleotides. In some embodiments, the one or more oligonucleotides are synthetic oligonucleotides. In some embodiments, the one or more oligonucleotides are adapters. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2. In some embodiments, the composition further comprises a divalent cation. In some embodiments, the composition further comprises double-stranded DNA.

[0042] A 16th aspect of the present disclosure is a method for processing a sample containing genomic material, the method comprising: (i) obtaining a tagmentation reaction mixture comprising a transposition system; (ii) introducing double-stranded DNA and a histone-like protein into the tagmentation reaction mixture to provide a fragmentation reaction mixture; and (iii) heating the fragmentation reaction mixture to a predetermined temperature for a predetermined time. In some embodiments, the double-stranded DNA and the histone-like protein are sequentially added to the tagmentation reaction mixture. In some embodiments, the double-stranded DNA and the histone-like protein are added simultaneously to the tagmentation reaction mixture. In some embodiments, the double-stranded DNA and the histone-like protein are first mixed together to form a DNA-histone-like protein solution, and then the DNA-histone-like protein solution is added to the tagmentation reaction mixture. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaeon. In some embodiments, the histone-like protein is an archaeal histone-like protein derived from either the genus Thermococcus or the genus Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1-2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1-2.

[0043] In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is about 5 ng / μL. In some embodiments, the ratio of the concentration of histone-like protein in the fragmentation reaction mixture to the concentration of double-stranded DNA in the tagmentation reaction mixture ranges from about 1:1 to about 4:1. In some embodiments, the ratio ranges from about 1.5:1 to about 3:1.

[0044] In some embodiments, the transposition system includes a transposase, a transposon, and an adapter. In some embodiments, the transposition system includes a hyperactive Tn5 transposase and a Tn5 transposase recognition site. In some embodiments, the transposition system includes a hyperactive Tn5 transposase, a Tn5 transposase recognition site, and one or more oligonucleotides. In some embodiments, the tagmentation reaction mixture further comprises a divalent cation selected from the group consisting of Co 2+ 、Mn 2+ 、Mg 2+ 、Cd 2+ 、and Ca 2+ .

Brief Description of the Drawings

[0045] The patent or application file includes at least one drawing created in color. Copies including the color drawings of this patent or patent application publication will be provided by the Patent Office upon request and payment of the necessary fees.

[0046] For a general understanding of the features of the present disclosure, reference is made to the drawings. In the drawings, like reference numerals are used throughout to identify like elements.

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DETAILED DESCRIPTION OF THE INVENTION

[0055] Detailed Description It should also be understood that, unless otherwise explicitly indicated to the contrary, in any method recited in the claims in this specification that includes a plurality of steps or acts, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0056] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" is defined inclusively such that "comprising A or B" means including A, B, or both A and B.

[0057] It should be understood that, as used in this specification and the claims, "or" has the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is to be interpreted as inclusive, i.e., including at least one of the number or list of elements, but also including a plurality and, if necessary, additional items not listed in the list. Only terms such as "only one of" or "exactly one of", or when used in the claims, "consisting of", etc., which are explicitly indicated to the contrary, refer to exactly one element of a number or list of elements. In general, the term "or" as used herein is to be interpreted as indicating an exclusive alternative (i.e., "one or the other but not both") only when preceded by exclusive terms such as "either", "only one of", "only one and exactly one of", etc. "Consisting essentially of", when used in the claims, shall have the ordinary meaning as used in the field of patent law.

[0058] Terms such as "comprising", "including", "having", etc. are used interchangeably and have the same meaning. Similarly, "comprises", "includes", "has", etc. are used interchangeably and have the same meaning. Specifically, each term is defined in accordance with the common United States patent law definition of "including,", and thus is interpreted as an open term meaning "at least" and not excluding additional features, limitations, aspects, etc. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus has at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c. Further, steps and processes may be outlined herein in a particular order, but one of ordinary skill in the art will recognize that the ordering of steps and processes may be different.

[0059] As used in the specification and claims of this specification, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of every element specifically listed within the list of elements, nor does it exclude any combinations of elements within the list of elements. This definition also allows for elements other than those specifically recited within the list of elements to which the phrase "at least one" refers to exist, if desired, regardless of whether or not they are related to those specifically recited elements. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") can, in one aspect, refer to at least one A, optionally including more than one, where B is absent (and optionally including elements other than B), in another aspect, can refer to at least one B, optionally including more than one, where A is absent (and optionally including elements other than A), and in yet another aspect, can refer to at least one A, optionally including more than one, and at least one B, optionally including more than one (and optionally including other elements), etc.

[0060] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0061] As used herein, the term "amplicon" refers to the product of a polynucleotide amplification reaction. That is, a cloned population of polynucleotides, which may be single-stranded or double-stranded, replicated from one or more starting sequences. The one or more starting sequences may be one or more copies of the same sequence or a mixture of various sequences. In some embodiments, an amplicon is formed by amplification of one starting sequence. An amplicon can be generated by various amplification reactions that produce a product consisting of a replica of one or more starting nucleic acids or target nucleic acids. In one aspect, the amplification reaction that produces the amplicon is "template-driven", and base pairing of any of the reactants, nucleotides or oligonucleotides has a complement in the template polynucleotide required for the production of the reaction product. In one aspect, the template-driven reaction is primer extension by a nucleic acid polymerase or oligonucleotide ligation by a nucleic acid ligase. Such reactions include, but are not limited to, polymerase chain reaction (PCR), linear polymerase reaction, nucleic acid sequence-based amplification (NASBA), rolling circle amplification, etc., each of which is hereby incorporated by reference in its entirety: Mullis et al., U.S. Patent Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al., U.S. Patent No. 5,210,015 ("taqman" probe for real-time PCR); Wittwer et al., U.S. Patent No. 6,174,670; Kacian et al., U.S. Patent No. 5,399,491 ("NASBA"); Lizardi, U.S. Patent No. 5,854,033; Aono et al., Japanese Patent Laid-Open No. 4-262799 (rolling circle amplification), etc. disclosed. In one aspect, the amplicons of the present invention are produced by PCR. The amplification reaction can be "real-time" amplification, such as "real-time PCR" or "real-time NASBA" described in, for example, Leone et al., Nucleic Acids Research, 26:2150-2155 (1998), when a detection chemistry is available that allows measurement of the reaction product as the amplification reaction proceeds.

[0062] As used herein, "amplification" refers to a process in which the copy number increases. Amplification can be a process in which replication occurs repeatedly over time to form multiple copies of a template. Amplification can result in an exponential or linear increase in the copy number as the amplification progresses. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade RCA, nucleic acid sequence-based amplification (NASBA), and the like. Also, amplification can utilize linear or circular templates. Amplification can be performed under any suitable temperature conditions using, for example, thermal cycling or isothermal conditions. Further, amplification can be carried out in an amplification mixture (or reagent mixture), which is any composition that can amplify a nucleic acid target if the nucleic acid target is present in the mixture. PCR amplification depends on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed between two or more temperature set points, such as a higher denaturation temperature and a lower annealing / extension temperature, or, in particular, between three or more temperature set points, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature. PCR can be performed using a thermostable polymerase such as Taq DNA polymerase. PCR generally results in an exponential increase in the amount of product amplicon over successive cycles.

[0063] As used herein, the terms "barcode array" or "molecular barcode" refer to a unique sequence of nucleotides that can be used to a) identify and / or track the source of polynucleotides during a reaction, b) count how many times an initial molecule has been sequenced, and c) pair sequence reads from different strands of the same molecule. Barcode arrays can vary widely in size and composition; the following references: Casbon (Nuc. Acids Res. 2011, 22 e81), Brenner, U.S. Patent No. 5,635,400; Brenner et al., Proc. Natl. Acad. Sci., 97:1665-1670 (2000); Shoemaker et al., Nature Genetics, 14:450-456 (1996); Morris et al., European patent publication 0799897A1; Wallace, U.S. Patent No. 5,981,179, etc. provide guidance for selecting a set of barcode arrays suitable for a particular embodiment. In certain embodiments, the barcode array can have a length in the range of 2 to 36 nucleotides, or 6 to 30 nucleotides, or 8 to 20 nucleotides.

[0064] As used herein, terms such as "biological sample", "tissue sample", "specimen" refer to any sample containing biomolecules (such as proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Other examples of organisms include mammals (such as veterinary animals like humans, cats, dogs, horses, cows, and pigs, as well as laboratory animals like mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections or needle biopsies of tissues), cell samples (such as cytological smear specimens like Pap smears or blood smears, or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (obtained by lysing cells and separating their components by centrifugation or the like). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucosa, tears, sweat, pus, biopsy tissue (such as obtained by surgical biopsy or needle biopsy), nipple aspirate, earwax, milk, vaginal fluid, saliva, rinse fluid (such as buccal swabs), or any material containing biomolecules derived from an initial biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample prepared from a tumor or a part thereof obtained from a subject (such as a homogenized or liquefied sample).

[0065] As used herein, the term "fragment" refers to a part of a larger polynucleotide molecule. Polynucleotides can be divided or fragmented into multiple segments by natural processes, such as in the case of cfDNA fragments that may naturally exist within a biological sample, or by in vitro manipulation. Samples can be fragmented by tagmentation.

[0066] As used herein, the term "mixture" refers to a combination of elements that are interspersed and not in a particular order. A mixture is heterogeneous and cannot be spatially separated into its various components. Examples of mixtures of elements include several different elements dissolved in the same aqueous solution and several different elements attached to a solid support at random positions (i.e., not in a particular order). A mixture cannot be addressed. By way of example, as is generally known in the art, an array of spatially separated surface-bound polynucleotides is not a mixture of surface-bound polynucleotides because the species of surface-bound polynucleotides are spatially distinct and the array is addressable.

[0067] As used herein, the term "next-generation sequencing" refers to sequencing technologies that have high-throughput sequencing as compared to traditional Sanger electrophoresis and capillary electrophoresis-based approaches, where the sequencing process is performed in parallel, generating, for example, thousands or millions of relatively small sequence reads at one time. Some examples of next-generation sequencing technologies include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. These technologies generate shorter reads (somewhere from about 25 to about 500 bp), but generate hundreds of thousands or millions of reads in a relatively short time. Examples of such sequencing devices available from Illumina (San Diego, Calif.) include, but are not limited to, iSEQ, MiniSEQ, MiSEQ, NextSEQ, and NovaSEQ. Illumina's next-generation sequencing technology is thought to enable rapid sequencing using clone amplification and sequencing by synthesis (SBS) chemistry. This process simultaneously identifies DNA bases while incorporating them into a nucleic acid strand. Each base emits a unique fluorescent signal when added to the growing strand, which is used to determine the order of the DNA sequence. Non-limiting examples of sequencing devices available from Thermo Fisher Scientific (Waltham, Mass.) include the Ion Personal Genome Machine™ (PGM™) System. Ion Torrent sequencing is thought to measure the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase. Non-limiting examples of sequencing devices available from Pacific Biosciences (Menlo Park, Calif.) include the PacBio Sequel System. A non-limiting example of a sequencing device available from Roche (Pleasanton, Calif.) is the Roche 454. Next-generation sequencing methods can also include nanopore sequencing methods. Generally, three nanopore sequencing approaches have been pursued.Strand sequencing, which is identified when the bases of DNA sequentially pass through a nanopore; exonuclease-based nanopore sequencing, in which nucleotides are enzymatically cleaved one by one from a DNA molecule and monitored as they are captured and passed through by the nanopore; and synthetic nanopore sequencing by synthesis (SBS) approach, in which distinguishable polymer tags are attached to nucleotides and registered in the nanopore during enzyme-catalyzed DNA synthesis. What is common to all these methods is that it is necessary to accurately control the reaction rate so that each base is determined in order. Strand sequencing requires a method to slow down the passage of DNA through the nanopore and decode multiple bases within the channel, and for this purpose, a ratchet approach using molecular motors has been developed. Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to ensure its capture and passage through the pore at a rate slow enough to obtain a valid ion current signal. Furthermore, both of these methods rely on the differences between the four natural bases, two relatively similar purines, and two similar pyrimidines. The nanopore SBS approach utilizes synthetic polymer labels attached to nucleotides that are specially designed to generate unique and easily distinguishable ion current blockade signatures for sequencing. In some embodiments, sequencing of nucleic acids by nanopore sequencing comprises preparing a nanopore sequencing complex and determining a polynucleotide sequence. Methods of preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017 / 0268052, as well as PCT Publications WO2014 / 074727, WO2006 / 028508, WO2012 / 083249, and WO / 2014 / 074727, the disclosures of which are hereby incorporated by reference in their entirety. In some embodiments, tagged nucleotides can be used in the determination of a polynucleotide sequence (see, e.g., PCT Publications WO / 2020 / 131759, WO / 2013 / 191793, and WO / 2015 / 148402).These disclosures are hereby incorporated by reference in their entirety). The analysis of data generated by sequencing generally involves software and / or statistical algorithms that perform various data conversions, such as converting signal emissions to base calls, and converting base calls to consensus sequences of nucleic acid templates. Such software, statistical algorithms, and the use of such software are described in detail in U.S. Patent Application Publication Nos. 2009 / 0024331, 2017 / 0044606, and PCT Publication No. WO / 2018 / 034745, which disclosures are hereby incorporated by reference in their entirety.

[0068] As used herein, the term "oligonucleotide" refers to a single-stranded polymer of nucleotides having about 2 to 200 nucleotides, up to 500 nucleotides in length. Oligonucleotides may be synthetic or enzymatically produced, and in some embodiments are 30 to 150 nucleotides in length. Oligonucleotides can contain ribonucleotide monomers (i.e., may be oligoribonucleotides) or deoxyribonucleotide monomers, or both ribonucleotide monomers and deoxyribonucleotide monomers. Oligonucleotides can be, for example, 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, or 150 to 200 nucleotides in length.

[0069] As used herein, the term "sequence," when used with respect to a nucleic acid molecule, refers to the order of nucleotides (or bases) in the nucleic acid molecule. Where different types of nucleotides are present in a nucleic acid molecule, the sequence includes the identification of the type of nucleotide (or base) at each position in the nucleic acid molecule. A sequence is a property of all or part of a nucleic acid molecule. This term can be used similarly to describe the order and positional identity of monomer units in other polymers, such as amino acid monomer units of a protein polymer.

[0070] As used herein, the term "sequencing" refers to the determination of the order and position of bases in a nucleic acid molecule. More specifically, the term "sequencing" refers to biochemical methods for determining the order of nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Sequencing, as the term is used herein, includes, but is not limited to, next-generation sequencing or any other sequencing method known to those of skill in the art, such as the chain termination method, rapid DNA sequencing, capillary electrophoresis, Maxam-Gilbert sequencing, dye terminator sequencing, or using any other up-to-date automated DNA sequencing apparatus.

[0071] As used herein, the term "tagmentation" or "tagmenting" refers to the process by which genomic DNA is fragmented, tagged with adapter sequences, and extended to fill in the gaps resulting from the fragmentation and tagging. More specifically, "tagmentation" refers to the modification of DNA by a transpososome complex containing a transposase enzyme complexed with an adapter containing transposon end sequences. Tagmentation results in the simultaneous fragmentation of DNA and ligation of adapters to the 5' ends of both strands of the double-stranded fragment. Tagmentation results in the simultaneous fragmentation of nucleic acids and ligation of adapters to the 5' ends of both strands of the double-stranded fragment. Following a purification step to remove the transposase enzyme, additional sequences may be added to the ends of the adapter fragments, for example, by PCR, ligation, or any other suitable method known to those of skill in the art.

[0072] As used herein, "transposition reaction" refers to a reaction in which one or more transposons are inserted into a target nucleic acid at a random or nearly random site. Essential components in the transposition reaction include a transposase and a DNA oligonucleotide that represent the nucleotide sequence of the transposon, which includes the transposed transposon sequence and its complement (i.e., the non-transposed transposon end sequence), as well as other components necessary to form a functional transposition or transposome complex. The DNA oligonucleotide may further include additional sequences (e.g., adapter or primer sequences) as needed or desired.

[0073] Summary

[0074] The present disclosure provides compositions and kits for the tagmentation of double-stranded DNA. In some embodiments, the compositions and kits for the tagmentation of double-stranded DNA include one or more histone-like proteins and / or one or more transposition systems. The present disclosure also provides methods for the tagmentation of double-stranded DNA in the presence of one or more histone-like proteins. Following the tagmentation of double-stranded DNA in the presence of one or more histone-like proteins, the tagmented DNA can be amplified and / or sequenced.

[0075] Compositions

[0076] One aspect of the present disclosure is a composition for use in a transposition reaction. In some embodiments, the composition includes one or more histone-like proteins and at least one additional component.

[0077] In some embodiments, the present disclosure provides a fragmentation composition comprising one or more histone-like proteins and one or more transposition systems. In some embodiments, the fragmentation composition comprises one or more histone-like proteins, one or more transposition systems, and one or more additional components such as divalent cations, buffers, polyols, DMSO, and the like.

[0078] In some embodiments, the present disclosure provides a fragmentation reaction mixture comprising one or more histone-like proteins, one or more transposition systems, and double-stranded DNA. In some embodiments, the present disclosure provides a fragmentation reaction mixture comprising one or more histone-like proteins, one or more transposition systems, double-stranded DNA, and one or more additional components such as divalent cations, buffers, polyols, DMSO, and the like.

[0079] In some embodiments, the present disclosure also provides a tagmentation reaction mixture comprising one or more transposition systems and one or more optional additional components such as divalent cations, buffers, polyols, DMSO, and the like.

[0080] In some embodiments, the present disclosure provides a DNA-histone-like protein solution comprising one or more histone-like proteins and double-stranded DNA.

[0081] The components of the fragmentation composition, the fragmentation reaction mixture, and the tagmentation reaction mixture are described herein.

[0082] Histone-like protein

[0083] As described above, the disclosed fragmented compositions and fragmented reaction mixtures each contain one or more histone-like proteins. Histone-like proteins (HLPs) are small basic bacterial proteins that associate with nucleotides and play a role in the regulation of DNA transactions such as DNA structure maintenance and replication, recombination / repair, and transcription. Architectural chromatin proteins are found in all domains of life. In eukaryotes and most archaeal lineages, histones are responsible for DNA packaging and compaction. Archaeal histone-like proteins show some homology to eukaryotic core histones in primary sequence, secondary, and tertiary structure.

[0084] Histone-like proteins are thought to act to wrap DNA by forming homo- and / or hetero-dimers and forming tetramers composed of dimers of dimers, forming what is called a nucleosome-like structure. Histone-like proteins exist as dimers in solutions without DNA and form stable tetramers in solutions containing double-stranded DNA. These proteins have been shown to compact DNA by forming these nucleosome-like structures that wrap DNA around the protein with a footprint of approximately 90 bp (in the case of the HphA homotetramer). When these histone-like proteins form nucleosome-like structures, they are thought to provide some nuclease protection by sterically inhibiting nucleases or blocking nuclease recognition sites.

[0085] In some embodiments, the histone-like protein is an archaeal histone-like protein. In other embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaeon. In some embodiments, the archaeal histone-like protein is derived from the genus Thermococcus. In other embodiments, the archaeal histone-like protein is derived from the genus Pyrococcus. In other embodiments, the archaeal histone-like protein is derived from Methanobacterium thermoautotrophicum. In other embodiments, the archaeal histone-like protein is derived from Methanothermus fervidus. In other embodiments, the archaeal histone-like protein is derived from Pyrococcus horikoshii. In other embodiments, the archaeal histone-like protein is derived from Pyrococcus horikoshii OT3.

[0086] Still other archaeal histone-like proteins may be derived from the phyla Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota (TACK), Diapherotrites, Pacearchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaeota (DPANN), and Asgard Archaea. Yet another archaeal histone-like protein may be derived from Asgard Archaea and candidate organisms from the phyla Bathyarchaeota, Woesearchaeota, Pacearchaeota, Aenigmarchaeota, Diapherotrites, Huberarchaea, and Micrarchaeota. Additional archaeal histone-like proteins are described in Henneman et al., “Structure and function of archaeal histones,” PLOS Genetics | https: / / doi.org / 10.1371 / journal.pgen.1007582 September 13, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0087] In some embodiments, the amino acid sequence encoding the histone-like protein has at least 80% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 85% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 86% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 87% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 88% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 89% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 90% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 91% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 92% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 93% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 94% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 95% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 96% sequence identity with any one of SEQ ID NOs: 1 and 2.In some embodiments, the amino acid sequence encoding the histone-like protein has at least 97% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 98% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has at least 99% sequence identity with any one of SEQ ID NOs: 1 and 2. In some embodiments, the amino acid sequence encoding the histone-like protein has any one of SEQ ID NOs: 1 and 2.

[0088] In some embodiments, the histone-like protein has at least 80% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 85% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 86% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 87% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 88% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 89% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 90% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 91% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 92% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 93% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 94% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 95% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 96% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 97% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 98% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has at least 99% sequence identity with SEQ ID NO: 3. In some embodiments, the histone-like protein has SEQ ID NO: 3.

[0089] In some embodiments, the histone-like protein has at least 80% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 85% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 86% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 87% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 88% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 89% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 90% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 91% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 92% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 93% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 94% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 95% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 96% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 97% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 98% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has at least 99% sequence identity with SEQ ID NO: 4. In some embodiments, the histone-like protein has SEQ ID NO: 4.

[0090] In some embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 1.5 ng / μL to about 50 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 2 ng / μL to about 40 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 2 ng / μL to about 35 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 2.5 ng / μL to about 35 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 2.5 ng / μL to about 30 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 2.5 ng / μL to about 25 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 2.5 ng / μL to about 20 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 3 ng / μL to about 30 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 3 ng / μL to about 25 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 3 ng / μL to about 20 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 4 ng / μL to about 35 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 4 ng / μL to about 30 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 4 ng / μL to about 25 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 4 ng / μL to about 20 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 5 ng / μL to about 35 ng / μL.In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 5 ng / μL to about 30 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 5 ng / μL to about 25 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 5 ng / μL to about 15 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 6 ng / μL to about 30 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 6 ng / μL to about 25 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 6 ng / μL to about 20 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 6 ng / μL to about 15 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 7 ng / μL to about 30 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 7 ng / μL to about 25 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 7 ng / μL to about 20 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 7 ng / μL to about 15 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 8 ng / μL to about 25 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 8 ng / μL to about 20 ng / μL. In other embodiments, the concentration of the histone-like protein in any composition or reaction mixture ranges from about 8 ng / μL to about 15 ng / μL.

[0091] In some embodiments, the histone-like protein is derived from Pyrococcus horikoshii OT3, and the concentration of the histone-like protein in any composition or reaction mixture is about 5 ng / μL. In some embodiments, the histone-like protein is derived from Pyrococcus horikoshii OT3, and the concentration of the histone-like protein in any composition or reaction mixture is about 7.5 ng / μL. In some embodiments, the histone-like protein is derived from Pyrococcus horikoshii OT3, and the concentration of the histone-like protein in any composition or reaction mixture is about 10 ng / μL. In some embodiments, the histone-like protein is derived from Pyrococcus horikoshii OT3, and the concentration of the histone-like protein in any composition or reaction mixture is about 15 ng / μL. In some embodiments, the histone-like protein is derived from Pyrococcus horikoshii OT3, and the concentration of the histone-like protein in any composition or reaction mixture is about 20 ng / μL.

[0092] In some embodiments, the histone-like protein has at least 85% identity with any one of SEQ ID NO: 1 and SEQ ID NO: 2, and the concentration of the histone-like protein in any composition or reaction mixture is about 5 ng / μL. In some embodiments, the histone-like protein has at least 85% identity with any one of SEQ ID NO: 1 and SEQ ID NO: 2, and the concentration of the histone-like protein in any composition or reaction mixture is about 7.5 ng / μL. In some embodiments, the histone-like protein has at least 85% identity with any one of SEQ ID NO: 1 and SEQ ID NO: 2, and the concentration of the histone-like protein in any composition or reaction mixture is about 10 ng / μL. In some embodiments, the histone-like protein has at least 85% identity with any one of SEQ ID NO: 1 and SEQ ID NO: 2, and the concentration of the histone-like protein in any composition or reaction mixture is about 15 ng / μL. In some embodiments, the histone-like protein has at least 85% identity with any one of SEQ ID NO: 1 and SEQ ID NO: 2, and the concentration of the histone-like protein in any composition or reaction mixture is about 20 ng / μL.

[0093] double-stranded DNA

[0094] In some embodiments, the compositions and / or reaction mixtures of the present disclosure comprise double-stranded DNA. In some embodiments, the double-stranded DNA is cDNA, ctDNA, or cfDNA. Of course, those skilled in the art will understand that any DNA suitable for use in the present disclosure can be initially converted from RNA using techniques known in the art.

[0095] Double-stranded DNA can be obtained from any source. For example, double-stranded DNA can be obtained from a single organism or from a population of nucleic acid molecules obtained from a natural source containing one or more organisms. Sources of nucleic acid molecules include, but are not limited to, organelles, cells, tissues, organs, organisms, single cells or single organelles. Cells that can be used as a source of the target nucleic acid molecule include prokaryotes (bacterial cells, e.g., Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma, Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrobacterium, Rhizobium, and Streptomyces); archaea such as crenarchaeota, nanoarchaeota or euryarchaeotia; eukaryotes such as fungi (e.g., yeast), plants, protozoa and other parasites, and animals (including insects (e.g., Drosophila spp.), nematodes (e.g., Caenorhabditis elegans) and mammals (e.g., rats, mice, monkeys, non-human primates and humans)).

[0096] In some embodiments, the double-stranded DNA is genomic DNA derived from a mammalian subject, such as a human patient. In some embodiments, the double-stranded DNA is derived from a tumor sample, such as a tumor sample derived from a human patient.

[0097] In some embodiments, the double-stranded DNA can enrich a specific sequence of interest prior to tagmentation. U.S. Patent Nos. 10,590,471, 10,900,068, 10,907,204, and 9,365,897; U.S. Patent Application Publication Nos. 2020 / 0048694 and 2020 / 0392483; and PCT Publication WO / 2012 / 108864 describe various methods for enriching a sequence of interest, the disclosures of which are hereby incorporated by reference in their entireties.

[0098] In some embodiments, the concentration of double-stranded DNA in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 0.5 ng / μL to about 15 ng / μL. In some embodiments, the concentration of double-stranded DNA in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 1 ng / μL to about 10 ng / μL. In some embodiments, the concentration of double-stranded DNA in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 3 ng / μL to about 7 ng / μL. In some embodiments, the concentration of double-stranded DNA in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 4 ng / μL to about 6 ng / μL. In some embodiments, the concentration of double-stranded DNA in any DNA-histone-like protein solution or fragmentation reaction mixture is about 4 ng / μL, about 5 ng / μL, about 6 ng / μL, or about 7 ng / μL.

[0099] In some embodiments, the ratio of the concentration of double-stranded DNA to the concentration of histone-like protein in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 0.25:1 to about 6:1. In some embodiments, the ratio of the concentration of double-stranded DNA to the concentration of histone-like protein in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 0.5:1 to about 5:1. In some embodiments, the ratio of the concentration of double-stranded DNA to the concentration of histone-like protein in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 1:1 to about 4:1. In some embodiments, the ratio of the concentration of double-stranded DNA to the concentration of histone-like protein in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 1.5:1 to about 3:1. In some embodiments, the ratio of the concentration of double-stranded DNA to the concentration of histone-like protein in any DNA-histone-like protein solution or fragmentation reaction mixture ranges from about 2:1 to about 1:2.

[0100] Transposition system

[0101] As described above, the fragmentation compositions and tagmentation reaction mixtures of the present disclosure each comprise one or more transposition systems. In some embodiments, any transposition system can be utilized in the fragmentation composition or tagmentation reaction mixture as long as the transposition system can fragment DNA. Suitable transposition systems are disclosed in Adey et al., “Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition,” Genome Biology 2010, 11:R119, the disclosure of which is incorporated herein by reference in its entirety.

[0102] In some embodiments, the transposition system comprises a transposase, a transposon or transposon DNA, and one or more oligonucleotides (e.g., barcodes, tags, adapters, etc.). In some embodiments, the transposase complexes with a transposon DNA comprising a double-stranded transposase binding site, and a first nucleic acid sequence comprising one or more of a tag, an adapter, or a barcode sequence and a priming site to form a transposase / transposon DNA complex. In some embodiments, the first nucleic acid sequence may be in the form of a single-stranded extension, or the first nucleic acid sequence may be in the form of a loop with each end connected to the corresponding strand of the double-stranded transposase binding site. In some embodiments, the transposase has the ability to bind to the transposon DNA and dimerize upon contact with each other to form a transposase / transposon DNA complex dimer called a transpososome. In some embodiments, the transpososome has the ability to bind to a target position along a double-stranded nucleic acid and form a complex comprising the transpososome and double-stranded genomic DNA. Such transposition systems are disclosed in U.S. Patent No. 10,894,980, U.S. Patent Application Publication No. 2018 / 0305683, and PCT Publication No. WO / 2015 / 089339, the disclosures of which are hereby incorporated by reference in their entireties.

[0103] In some embodiments, the transposase complexed with transposon DNA comprises a dimer of the transposase and a pair of adapters (see U.S. Patent Application Publication No. 2018 / 0305683, the disclosure of which is incorporated herein by reference in its entirety). The term "adapter" refers to a nucleic acid that can be linked to at least one strand of a double-stranded nucleic acid molecule (e.g., double-stranded DNA) via a transposase-mediated reaction. The adapter may be at least partially double-stranded and may be 30 to 150 bases in length, such as 40 to 120 bases in length. In other embodiments, the transposase complex comprises a transposase loaded with two adapter molecules each containing a recognition sequence of the transposase at one end. In still other embodiments, the transposase complexed with transposon DNA comprises a dimer of a modified transposase Tn5 and a pair of 19-base pair Tn-5 binding double-stranded DNA oligonucleotides containing a transposase binding sequence (mosaic end) or an inverted repeat sequence. In yet further embodiments, the transposition system comprises at least one first oligonucleotide comprising at least one double-stranded portion, the double-stranded portion comprising at least one first recognition end sequence; at least one second oligonucleotide comprising at least one double-stranded portion, the double-stranded portion comprising at least one second recognition end sequence; and a transposase (see PCT Publication No. WO / 2015 / 089339, the disclosure of which is incorporated herein by reference in its entirety).

[0104] In other embodiments, the transposition system includes a transposase, a transposon end composition, and / or an adapter. As used herein, the term "transposase" refers to an enzyme that forms a functional complex with a transposon end-containing composition (e.g., a transposon, a transposon end, a transposon end composition) and catalyzes the insertion or transposition of the transposon end-containing composition into double-stranded target DNA incubated in an in vitro transposition reaction. As used herein, the phrase "transposon end composition" refers to a composition that includes a transposon end (i.e., the minimal double-stranded DNA segment that can act with a transposase to cause a transposition reaction) and optionally one or more additional sequences. For example, a transposon end with a tag attached is a "transposon end composition." In some embodiments, the transposon end composition includes two transposon end oligonucleotides that together represent the sequence of the transposon end and in which one or both strands include additional sequences, a "transferred transposon end oligonucleotide" or "transcribed strand" and a "non-transcribed strand end oligonucleotide," or "non-transcribed strand." Such transposition systems are described in U.S. Patent Nos. 11,118,175, 10,815,478, and 10,184,122, the disclosures of which are incorporated herein by reference in their entirety.

[0105] In some embodiments, the transposition system comprises TnAa. In some embodiments, the transposition system can utilize a highly active Tn5 transposase and Tn5 transposase recognition sites (Goryshin and Reznikoff, J. Biol. Chem., 273:7367 (1998)), or a MuA transposase and a Mu transposase recognition site comprising R1 and R2 end sequences (Mizuuchi, K., Cell, 35:785, 1983; Savilahti, H, et al., EMBO J., 14:4893, 1995).In some other embodiments, the transposition system can utilize Staphylococcus aureus Tn552 (Colegio et al., J. Bacteriol., 183:2384-8, 2001; Kirby C et al., Mol. Microbiol., 43:173-86, 2002), Ty1 (Devine and Boeke, Nucleic Acids Res., 22:3765-72, 1994 and WO 95 / 23875), transposon Tn7 (Craig, N L, Science.271:1512, 1996; Craig, N L, Review in: Curr Top Microbiol Immunol., 204:27-48, 1996), Tn / O and IS10 (Kleckner N, et al., Curr Top Microbiol Immunol., 204:49-82, 1996), mariner transposase (Lampe D J, et al., EMBO J., 15:5470-9, 1996), Tc1 (Plasterk R H, Curr.Topics Microbiol.Immunol., 204:125-43, 1996), P element (Gloor, G B, Methods Mol.Biol., 260:97-114, 2004), Tn3 (Ichikawa and Ohtsubo, J Biol.Chem.265:18829-32, 1990), bacterial insertion sequences (Ohtsubo and Sekine, Curr.Top.Microbiol.Immunol.204:1-26, 1996), retroviruses (Brown, et al., Proc Natl Acad Sci USA, 86:2525-9, 1989), and yeast retrotransposons (Boeke and Corces, Annu Rev Microbiol.43:403-34, 1989). Further alternative transposition systems can include ISS, Tn10, Tn903, IS911, and engineered versions of transposase family enzymes (Zhang et al., (2009) PLoS Genet.5:e1000689.Epub 2009 Oct.16; Wilson C. et al. (2007) J.Microbiol.Methods 71:332-5).

[0106] Further useful transposition systems include the Tn3 transposition system (see Maekawa, T., Yanagihara, K., and Ohtsubo, E. (1996), A cell-free system of Tn3 transposition and transposition immunity, Genes Cells 1, 1007-1016), the Tn10 transposition system (see Chalmers, R., Sewitz, S., Lipkow, K., and Crellin, P. (2000), Complete nucleotide sequence of Tn10, J. Bacteriol 182, 2970-2972), the Piggybac transposase tool for genome engineering (see Li, X., Burnight, E. R., Cooney, A. L, Malani, N., Brady, T., Sander, J. D., Staber, J., Wheelan, S. J., Joung, J. K., McCray, P. B., Jr., et al. (2013), PiggyBac transposase tools for genome engineering, Proc. Natl. Acad. Sci. USA 110, E2279-2287), the Sleeping beauty transposition system (see Ivies, Z., Hackett, P. B., Piasterk, R. H., and Izsvak, Z. (1997), Molecular reconstruction of Sleeping Beauty, the Tc1-like transposon from fish, and its transposition in human cells, Cell 91, 501-510), and the Tol2 transposition system (see Kawakami, K. (2007), Tol2: a versatile gene transfer vector in vertebrates, Genome Biol. 8 Suppl. 1, S7).As yet another suitable transposition system, for example, there is one provided by Illumina in the NEXTERA DNA or NEXTERA DNA Flex library preparation kit. Further, additional transposition systems or components thereof are disclosed in U.S. Patent Nos. 7,608,434, 7,083,980, 5,965,443, and 5,925,545, the disclosures of which are incorporated herein by reference in their entireties.

[0107] In some embodiments, the transposase included in any transposition system has 95% sequence identity with SEQ ID NO: 5 (TnAa). In some embodiments, the transposase has 96% sequence identity with SEQ ID NO: 5. In some embodiments, the transposase has 97% sequence identity with SEQ ID NO: 5. In some embodiments, the transposase has 98% sequence identity with SEQ ID NO: 5. In some embodiments, the transposase has 99% sequence identity with SEQ ID NO: 5. In some embodiments, the transposase has SEQ ID NO: 5. In some embodiments, the transposase has at least 95% sequence identity with SEQ ID NO: 6. In some embodiments, the transposase has at least 97% sequence identity with SEQ ID NO: 6. In some embodiments, the transposase has at least 99% sequence identity with SEQ ID NO: 6. In some embodiments, the transposase has SEQ ID NO: 6.

[0108] In some embodiments, the concentration of the transposition system in any composition or reaction mixture ranges from about 100 ng / μL to about 400 ng / μL. In some embodiments, the concentration of the transposition system in any composition or reaction mixture ranges from about 100 ng / μL to about 250 ng / μL. In some embodiments, the concentration of the transposition system in any composition or reaction mixture ranges from about 120 ng / μL to about 230 ng / μL. In some embodiments, the concentration of the transposition system in any composition or reaction mixture ranges from about 140 ng / μL to about 210 ng / μL. In some embodiments, the concentration of the transposition system in any composition or reaction mixture ranges from about 150 ng / μL to about 200 ng / μL. In some embodiments, the concentration of the transposition system in any composition or reaction mixture ranges from about 160 ng / μL to about 190 ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is about 160 ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is about 170 ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is about 175 ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is about 180 ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is about 185 ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is about 190 ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is about 200 ng / μL.

[0109] divalent cation

[0110] In some embodiments, the fragmentation composition, fragmentation reaction mixture, and / or tagging reaction mixture may contain one or more divalent cations. In some embodiments, the divalent cation is Co2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ selected from 。

[0111] In some embodiments, either the composition or the reaction mixture of the present disclosure can include a divalent cation concentration of at least about 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM. In some embodiments, either the composition or the reaction mixture of the present disclosure can have a CoCl2 concentration of at least about 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM. In some embodiments, either the composition or the reaction mixture of the present disclosure can have a MnCl2 concentration of at least about 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM. In some embodiments, either the composition or the reaction mixture of the present disclosure can have a MgCl2 concentration of at least about 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM. In some embodiments, either the composition or the reaction mixture of the present disclosure can have a CdCl2 concentration of at least about 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM.In some embodiments, either the composition or the reaction mixture of the present disclosure can have a concentration of CaCl2 that is at least about 0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM.

[0112] Buffer

[0113] In some embodiments, either the composition or the reaction mixture of the present disclosure contains one or more buffers. Non-limiting examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), and combinations thereof. In other embodiments, the buffer can consist of tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or combinations thereof.

[0114] Additional components

[0115] In some embodiments, either the composition or the reaction mixture of the present disclosure comprises a polyol. Suitable polyols include 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 2-methyl-1,3-propanediol, 2-methyl-1,2-propanediol, 2,2-dimethyl-1,3-propanediol, 2,2-diethyl-1,3-propanediol, 2-methyl-2-propyl-1,3-propanediol, 2-butyl-2-ethyl-1,3-propanediol, dihydroxyacetone, 2,2-dibutyl-1,3-propanediol, 3-methoxy-1,3-propanediol, 3-methoxy-1,2-propanediol, 3-methoxy-2,3-propanediol, 2-methoxymethyl-1,3-propanediol, 3-ethoxy-1,3-propanediol, 3-ethoxy-1,2-propanediol, 3-ethoxy-2,3-propanediol, 3-allyloxy-1,2-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 2,3-dimethyl-2,3-butanediol, 3,3-dimethyl-1,2-butanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,5-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 2-methyl-2,4-pentanediol, 2,4-dimethyl-2,4-pentanediol, 2,2,4-trimethyl-1,3-pentanediol, 1,2-hexanediol, 1,3-hexanediol, 1,4-hexanediol, 1,5-hexanediol, 1,6-hexanediol, 2,3-hexanediol, 2,4-hexanediol, 2,5-hexanediol, 3,4-hexanediol, 2,5-dimethyl-2,5-hexanediol, 2-ethyl-1,3-hexanediol, 1,2-heptanediol, 1,3-heptanediol, 1,4-heptanediol, 1,5-heptanediol, 1,6-heptanediol, 1,7-heptanediol, 1,8-octanediol, 1,2-octanediol, 1,3-octanediol, 1,4-octanediol, 1,5-octanediol, 1,6-octanediol, 1,7-octanediol, 1,2-nonanediol, 1,9-Nonadiol, 1,10-Decanediol, 1,2-Decanediol, 1,2-Undecanediol, 1,11-Undecanediol, 1,12-Dodecanediol, 1,2-Dodecanediol, Diethylene Glycol, Dipropylene Glycol, Triethylene Glycol, Tripropylene Glycol, Tetraethylene Glycol, Tetrapropylene Glycol, Pentaethylene Glycol, Pentapropylene Glycol, Hexaethylene Glycol, Hexapropylene Glycol, Heptaethylene Glycol, Heptapropylene Glycol, Octaethylene Glycol, Octapropylene Glycol, Nonaethylene Glycol, Nonapropylene Glycol, Decaethylene Glycol, Decapropylene Glycol, cis- or trans-1,2-Cyclopentanediol, cis- or trans-1,3-Cyclopentanediol, cis- or trans-1,2-Cyclohexanediol, cis- or trans-1,3-Cyclohexanediol, cis- or trans-1,4-Cyclohexanediol, cis- or trans-1,2-Cyclohexanediol, cis- or trans-1,3-Cycloheptanediol, cis- or trans-1,4-Cycloheptanediol, 1,2,3-Cyclopentanetriol, 1,2,4-Cyclopentanetriol, 1,2,3-Cyclohexanetriol, 1,2,4-Cyclohexanetriol, 1,2,3-Cycloheptanetriol, 1,2,4-Cycloheptanetriol, 1,2,3-Propanetriol, 3-Ethyl-2-hydroxymethyl-1,3-propanediol, 2-Hydroxymethyl-2-methyl-1,3-propanediol, 1,2,3-Butanetriol, 1,2,4-Butanetriol, 2-Methyl-1,2,3-butanetriol, 2-Methyl-1,2,4-butanetriol, 1,2,3-Pentanetriol, 1,2,4-Pentanetriol, 1,2,5-Pentanetriol, 2,3,4-Pentanetriol, 1,3,5-Pentanetriol, 3-Methyl-1,3,5-pentanetriol, 1,2,3-Hexanetriol, 1,2,4-Hexanetriol, 1,2,5-Hexanetriol, 1,2,6-Hexanetriol, 2,3,4-Hexanetriol, 2,3,5-Hexanetriol, 1,2,3-Heptanetriol, 1,2,7-heptanetriol, 1,2,3-octanetriol, 1,2,8-octanetriol, 1,2,3-nonanetriol, 1,2,9-nonanetriol, 1,2,3-decanetriol, 1,2,10-decanetriol, 1,2,3-undecanetriol, 1,2,11-undecanetriol, 1,2,3-dodecanetriol, 1,1,12-dodecanetriol, 2,2-bis(hydroxymethyl)-1,3-propanediol, 1,2,3,4-butanetetraol, 1,2,3,4-pentanetetraol, 1,2,3,5-pentanetetraol, 1,2,3,4-hexanetetraol, 1,2,3,6-hexanetetraol, 1,2,3,4-heptanetetraol, 1,2,3,7-heptanetetraol, 1,2,3,4-octanetetraol, 1,2,3,8-octanetetraol, 1,2,3,4-nonanetetraol, 1,2,3,9-nonanetetraol, 1,2,3,4-decanetetraol, 1,2,3,10-decanetetraol, trimethylolpropane, pentaerythritol, sugar alcohols such as mannitol, sorbitol or arabitol, hexanehexol, 1,2,3,4,5-pentanepentol and 1,2,3,4,5,6-hexanehexol. In some embodiments, the polyol is 1,2,3-propanetriol (also known as glycerol).

[0116] In some embodiments, either the composition or the reaction mixture of the present disclosure contains dimethyl sulfoxide.

[0117] In some embodiments, either the composition or the reaction mixture of the present disclosure contains one or more salts or surfactants.

[0118] Method for Tagmentation in the Presence of Histone-like Protein

[0119] The present disclosure also provides a method of tagging DNA in the presence of one or more histone-like proteins. Generally, the method includes forming a fragmentation reaction mixture and heating the fragmentation reaction mixture at a predetermined temperature for a predetermined period of time. Various embodiments of this general method are described herein.

[0120] Referring to FIG. 1, a sample is first obtained (step 101). In some embodiments, the sample comprises double-stranded DNA. In some embodiments, the double-stranded DNA is genomic DNA. In some embodiments, the double-stranded DNA is genomic DNA derived from a mammalian subject, such as a human patient. In some embodiments, the double-stranded DNA is derived from a tumor sample. In some embodiments, the double-stranded DNA is cDNA or ctDNA.

[0121] In some embodiments, a fragmentation composition is added to the obtained sample (step 102) to obtain a fragmentation reaction mixture. As described herein, the fragmentation composition can include one or more histone-like proteins, one or more transposition systems, and one or more optional additional components. In some embodiments, the obtained sample is allowed time to mix with the fragmentation composition before being heated. In some embodiments, the obtained sample is mixed with the fragmentation composition for about 30 seconds, about 1 minute, about 2 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 10 minutes, about 20 minutes, or about 30 minutes before being heated.

[0122] Subsequently, the fragmented reaction mixture is heated at a predetermined temperature for a predetermined amount of time (Step 103), i.e., the tagging reaction is carried out by heating the fragmented reaction mixture. In some embodiments, the tagging reaction can be carried out at a temperature in the range of about 25°C to about 70°C, about 37°C to about 65°C, about 50°C to about 65°C, or about 50°C to about 60°C. In some embodiments, the tagging reaction can be carried out at a temperature of about 37°C, about 40°C, about 45°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, or about 65°C. In some embodiments, the tagging reaction can be carried out for a time in the range of about 30 seconds to about 10 minutes; about 1 minute to about 8 minutes; about 2 minutes to about 8 minutes; about 3 minutes to about 7 minutes; or about 4 minutes to about 6 minutes. In some embodiments, the tagging reaction can be carried out for about 2 minutes; about 3 minutes; about 4 minutes; about 5 minutes; or about 6 minutes. Methods and additional reaction conditions for tagging DNA using a transposition system are described in U.S. Patent No. 9,080,211 and U.S. Patent Application Publication No. 2010 / 0120098, the disclosures of which are hereby incorporated by reference in their entirety.

[0123] Next, the tagged DNA is isolated from the fragmented reaction mixture (Step 104). In some embodiments, the isolation of the tagged DNA includes (a) capturing the DNA fragments generated using a capture probe onto beads; (b) flushing impurities and untargeted fragments from the reaction mixture; and (c) eluting or releasing the captured DNA fragments from the beads or the capture probe.

[0124] In some embodiments, the tagged DNA has an average fragment size in the range of about 200 bp to about 400 bp, about 220 bp to about 360 bp, about 240 bp to about 330 bp, or about 250 bp to about 300 bp.

[0125] Other methods of tagging DNA are shown in Figure 2. In this embodiment, a tagging reaction mixture is first obtained (step 201). Subsequently, double-stranded DNA and histone-like proteins are introduced into the tagging reaction mixture to form a fragmentation reaction mixture (step 202). In some embodiments, the double-stranded DNA and histone-like proteins are added sequentially (see Figure 3, i.e., steps 302 and 303 which can be performed in any order). In other embodiments, the double-stranded DNA and histone-like proteins are added simultaneously. In yet other embodiments, the double-stranded DNA and histone-like proteins are first combined to form a DNA-histone-like protein solution, which is then added to the tagging reaction mixture to form a fragmentation reaction mixture. Next, the fragmentation reaction mixture is heated at a predetermined temperature for a predetermined time (step 103). Following tagging, the tagged DNA is isolated (step 104). In some embodiments, the tagged DNA is "cleaned up" prior to amplification to remove impurities. In some embodiments, the "cleanup" utilizes functionalized beads such as KAPA PureBeads (KAPA Biosystems, Inc. (Wilmington, Massachusetts)).

[0126] Following tagging and isolation of the tagged DNA, the tagged DNA can optionally be amplified (step 401) and / or sequenced (step 402). In some embodiments, the sequencing is performed using next-generation sequencing.

[0127] Kit Components

[0128] The present disclosure also provides a kit comprising any of the compositions or reaction mixtures described herein. In some embodiments, the kits disclosed herein may optionally include other components including, but not limited to, reagents for performing a polymerase chain reaction (e.g., PCR primers, polymerases, buffers, nucleotides, etc.). The various components of the kits of the present disclosure may be present in separate containers or, optionally, certain compatible components may be pre-combined in a single container as desired.

[0129] In some embodiments, any of the kits of the present disclosure may further comprise one or more reagents for performing a polymerase chain reaction (PCR). In some embodiments, the PCR reagents include deoxynucleoside triphosphates (dNTPs), particularly all four naturally occurring deoxynucleoside triphosphates (dNTPs). In some embodiments, the PCR reagents include deoxyribonucleoside triphosphate molecules that include all of dATP, dCTP, dGTP, and dTTP. In some embodiments, the PCR reagents also include compounds useful for assisting the activity of nucleic acid polymerases. For example, in some embodiments, the PCR reagents include divalent cations, such as magnesium ions. In some embodiments, the magnesium ions are provided in the form of magnesium chloride, magnesium acetate, or magnesium sulfate. In some embodiments, the PCR reagents further include a buffer or buffer solution that includes any of the buffers listed herein.

[0130] In some embodiments, any of the kits of the present disclosure may further comprise at least one polymerase, modified polymerase, or thermostable polymerase. As used herein, the term "polymerase" refers to an enzyme that performs the synthesis of polynucleotides directed towards a template. DNA polymerases can add free nucleotides only to the 3' end of the newly formed strand. Thereby, the newly formed strand extends in the 5'-3' direction. Known DNA polymerases cannot initiate a new strand (de novo). DNA polymerases can add nucleotides only to an existing 3'-OH group and thus require a primer that can add the first nucleotide. Non-limiting examples of polymerases include prokaryotic DNA polymerases (e.g., Pol I, Pol II, Pol III, Pol IV, and Pol V), eukaryotic DNA polymerases, archaeal DNA polymerases, telomerase, reverse transcriptase, and RNA polymerase. Reverse transcriptase is an RNA-dependent DNA polymerase that synthesizes DNA from an RNA template. The reverse transcriptase family includes both the functionality of DNA polymerase and the functionality of RNaseH, which degrades RNA base-paired to DNA. RNA polymerase is an enzyme that synthesizes RNA using DNA as a template in the process of gene transcription. RNA polymerase polymerizes ribonucleotides at the 3' end of the RNA transcript.

[0131] In some embodiments, suitable polymerases include archaebacteria (e.g., Thermococcus litoralis (Vent, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp. KOD strain (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811), Aeropyrum pernix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus sp. GE8 (GenBank: CAC12850), Thermococcus sp.)JDF-3 (GenBank: AX135456; WO0132887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23 (GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii (GenBank: Q58295), Desulforococcus species TOK, Desulforococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025) and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579), thermophilic bacteria Thermus species (e.g., flavus, ruber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp., 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T.It may be derived from Thermococcus zilligii, T. sp., GT, P. sp. GB-D, KOD, Pfu, Thermococcus gorgonarius, Thermococcus zilligii, Thermococcus litoralis, and Thermococcus sp. 9N-7 polymerase.

[0132] As used herein, the term "modified DNA polymerase" refers to a DNA polymerase that is derived from a different (i.e., parental) DNA polymerase and contains one or more amino acid changes (e.g., amino acid substitutions, deletions, or insertions) compared to the parental DNA polymerase. In some embodiments, the modified DNA polymerases of the disclosure are derived from or modified from naturally occurring or wild-type DNA polymerases. In some embodiments, the modified DNA polymerases of the disclosure are derived from or modified from recombinant or engineered DNA polymerases including, but not limited to, chimeric DNA polymerases, fusion DNA polymerases, or other modified DNA polymerases. Typically, the modified DNA polymerase has at least one changed phenotype compared to the parental polymerase. Examples of modified polymerases are described in U.S. Patent Application Publication No. 2016 / 0222363, the disclosure of which is incorporated herein by reference in its entirety.

[0133] As used herein, the term "thermostable polymerase" refers to an enzyme that is stable to heat, is heat resistant, and retains sufficient activity to subsequently perform a polynucleotide extension reaction when exposed to a high temperature for a time necessary to denature double-stranded nucleic acid and is not irreversibly denatured (inactivated). Heating conditions necessary for nucleic acid denaturation are known in the art and are exemplified, for example, in U.S. Patent Nos. 4,683,202, 4,683,195, and 4,965,188, which are incorporated herein by reference. As used herein, a thermostable polymerase is suitable for use in temperature cycling reactions such as polymerase chain reaction ("PCR"), primer extension reactions, or terminal modification (e.g., terminal transferase, digestion or polish) reactions. Irreversible denaturation for the purposes of this specification refers to the permanent and complete loss of enzyme activity. In a thermostable polymerase, enzyme activity refers to the catalysis of a combination of nucleotides in a manner appropriate to form a polynucleotide extension product that is complementary to the template nucleic acid strand. Examples of thermostable DNA polymerases derived from thermophilic bacteria include, for example, DNA polymerases derived from Thermotoga maritima, Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermus sps17, Thermus sp Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, Thermosipho africanus, and other thermostable DNA polymerases disclosed above.

[0134] In some embodiments, the polymerase can be a modified naturally occurring type A polymerase. Further embodiments of the invention generally relate to methods in which a modified type A polymerase, such as in primer extension, end modification (e.g., terminal transferase, digestion or polishing), or amplification reactions, can be selected from any species of the genus Meiothermus, Thermotoga, or Thermomicrobium. Another embodiment of the invention generally relates to methods in which the polymerase can be isolated from any of Thermus aquaticus (Taq), Thermus thermophilus, Thermus caldophilus, or Thermus filiformis, such as in primer extension, end modification (e.g., terminal transferase, digestion or polishing), or amplification reactions. Further embodiments of the invention generally include methods in which a modified type A polymerase can be isolated from Bacillus stearothermophilus, Sphaerobacter thermophilus, Dictoglomus thermophilum, or Escherichia coli, such as in primer extension, end modification (e.g., terminal transferase, digestion or polishing), or amplification reactions. In another embodiment, the invention generally relates to methods in which a modified type A polymerase, such as in primer extension, end modification (e.g., terminal transferase, digestion or polishing), or amplification reactions, can be the mutant Taq-E507K polymerase. Another embodiment of the invention generally relates to methods in which a thermostable polymerase can be used to amplify a target nucleic acid.

[0135] In some embodiments, any of the kits of the present disclosure may further include a ligase. In some embodiments, the ligase is a DNA ligase. In some embodiments, the ligase is a thermostable single-stranded RNA or DNA ligase, such as Thermophage ligase or a derivative thereof, such as Circligase™ and Circligase™ II (Epicentre Tech., Madison, Wise.). In other embodiments, the ligase is a T4 ligase.

[0136] In addition to the above components, the subject kit may further include instructions for using the components of the kit to perform the subject method, i.e., instructions for library preparation. Instructions for performing the subject method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. Accordingly, the instructions may be present in the kit as an accompanying document, on a label of the kit's container or its components (i.e., associated with the package or subpackage). In other embodiments, the instructions are present as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means are provided for obtaining the instructions from a remote source, e.g., via the Internet. An example of this embodiment is a kit that includes a web address from which the instructions can be viewed and / or downloaded.

[0137] In some embodiments, any one of the kits of the present disclosure may include a sequencing device, such as a sequencing device for "next-generation sequencing". The term "next-generation sequencing" refers to sequencing technologies that have high-throughput sequencing compared to conventional Sanger electrophoresis and capillary electrophoresis-based approaches, where the sequencing process is performed in parallel and generates, for example, thousands or millions of relatively small sequence reads at once. Some examples of next-generation sequencing technologies include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. These technologies generate shorter reads (somewhere from about 25 to about 500 bp), but generate hundreds of thousands or millions of reads in a relatively short time.

[0138] Examples of such sequencing devices available from Illumina (San Diego, California) include, but are not limited to, iSEQ, MiniSEQ, MiSEQ, NextSEQ, and NovaSEQ. Illumina's next-generation sequencing technology is thought to enable rapid sequencing using clonal amplification and sequencing by synthesis (SBS) chemistry. This process identifies DNA bases while incorporating them into a nucleic acid strand simultaneously. Each base emits a unique fluorescent signal when added to the growing strand, which is used to determine the order of the DNA sequence.

[0139] Non-limiting examples of sequencing devices available from ThermoFisher Scientific (Waltham, Massachusetts) include the Ion Personal Genome Machine (trademark) (PGM (trademark)) system. Ion Torrent sequencing is thought to measure the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase. Non-limiting examples of sequencing devices available from Pacific Biosciences (Menlo Park, California) include the PacBio Sequel System. A non-limiting example of a sequencing device available from Roche (Pleasanton, California) is the Roche 454.

[0140] Next-generation sequencing methods can include nanopore sequencing methods. Generally, three nanopore sequencing approaches have been pursued. Strand sequencing, which is identified when DNA bases sequentially pass through a nanopore, exonuclease-based nanopore sequencing, in which nucleotides are enzymatically cleaved one by one from a DNA molecule and monitored as they are captured and passed through by a nanopore, and synthetic nanopore sequencing (SBS) approaches in which distinguishable polymer tags are attached to nucleotides and registered in a nanopore during enzyme-catalyzed DNA synthesis. What is common to all of these methods is the need to precisely control the reaction rate so that each base is determined in order.

[0141] Strand sequencing requires a method that slows the passage of DNA through a nanopore and decodes multiple bases within the channel. For this purpose, a ratchet approach using a molecular motor has been developed. Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to ensure its capture and passage through the pore at a rate slow enough to obtain a valid ion current signal. Furthermore, both of these methods rely on the differences between the four natural bases, two relatively similar purines, and two similar pyrimidines. The nanopore SBS approach utilizes synthetic polymer labels attached to nucleotides that are specially designed to generate unique and readily distinguishable ion current blockade signatures for sequencing. In some embodiments, sequencing of nucleic acids by nanopore sequencing comprises preparing a nanopore sequencing complex and determining a polynucleotide sequence. Methods of preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017 / 0268052, as well as International Publication Nos. 2014 / 074727, 2006 / 028508, 2012 / 083249, and 2014 / 074727, the disclosures of which are hereby incorporated by reference in their entirety. In some embodiments, tagged nucleotides can be used in the determination of polynucleotide sequences (see, e.g., PCT Publications WO / 2020 / 131759 Pamphlet, WO / 2013 / 191793 Pamphlet, and WO / 2015 / 148402 Pamphlet, the disclosures of which are hereby incorporated by reference in their entirety).

[0142] In some embodiments, any one of the kits of the present disclosure may include software for analyzing the obtained sequencing data. Analysis of data generated by sequencing generally involves software and / or statistical algorithms that perform various data conversions, such as converting signal emissions to base calls, converting base calls to consensus sequences of nucleic acid templates, etc. Such software, statistical algorithms, and the use of such software are described in detail in U.S. Patent Application Publication Nos. 2009 / 0024331, 2017 / 0044606, and PCT Publication No. WO / 2018 / 034745, the disclosures of which are hereby incorporated by reference in their entirety.

Example

[0143] Tagmentation of DNA in the presence of histone-like protein was demonstrated using 10 ng of Escherichia coli DNA (mg1655) and transposase TnAa (Tn5L1) at a stock concentration of 180 ng / μL. This experiment was designed to demonstrate that larger fragments can be obtained by adding histone-like protein without using dilution of the transposition system concentration to control fragment size. A tagmentation reaction setup containing a total input of 2.5 ng to 80 ng of the transposition system (TnAa) resulted in a similar range of library sizes compared to tagmentation in the presence of histone-like protein (compare FIGS. 5 and 6).

[0144] The components of the reaction setup are shown below.

[0145] 10 ng of Escherichia coli DNA (mg1655) was tagmented and sequencing libraries were prepared at the following TnAa (Tn5L1) enzyme concentrations: 2.5 ng (0.625 ng / μL), 5 ng (1.25 ng / μL), 10 ng (2.5 ng / μL), 20 ng (5 ng / μL), 30 ng (7.5 ng / μL), and 80 ng (20 ng / μL). The TnAa transposase included in the transposition system has SEQ ID NO: 5.

[0146] Tagmentation reaction mixture [Table 1]

[0147] LMW buffer (2×): [Table 2]

[0148] Dilution buffer [Table 3]

[0149] Storage buffer [Table 4]

[0150] In the presence of histone-like protein (HphA), 10 ng of Escherichia coli DNA (mg1655) was tagmented. The fragmentation reaction mixture contained the transposition system at a concentration of 180 ng / μL, and the histone-like protein was included in amounts of 40 ng (20 ng / μL), 30 ng (15 ng / μL), 20 ng (10 ng / μL), 15 ng (7.5 ng / μL), 5 ng (2.5 ng / μL), 0 ng. In each experiment, the DNA concentration was kept constant at 5 ng / μL.

[0151] Fragmentation reaction mixture [Table 5]

[0152] The tagmentation reaction conditions were as follows.

[0153] 5 minutes at 52 °C

[0154] 5 μL of stop solution was added (0.25% SDS) [What else is included in the stop solution?]

[0155] 0.8x Kappa PureBeads cleanup

[0156] Elute in 22 μL of 10 mM Tris

[0157] Amplification was performed after preparation of tagged DNA using the following parameters.

Table 6

Table 7

[0158] The samples were then sequenced on an Illumina MiniSeq using the MiniSeq System Mid-Output Kit (2×150 bp). Sequence reads were quality filtered, sampled, and evaluated based on insert size and sequence bias at the insertion site.

[0159] The average insert size of the tagmentation library increased with increasing amounts of histone-like protein added (see Figure 7). The insert size reached a plateau at 300 bp when 20 ng - 40 ng of histone-like protein was added (see Figure 7). The bias at the start site was not affected by the amount of histone added to the tagmentation reaction (data not shown). The average insert size of the library decreased with increasing amounts of TnAa enzyme (see Figure 8). As a result, it was shown that a stock concentration of transposase enzyme (180 ng / μL) with an average insert size of up to 300 bp can be used to obtain larger insert sizes.

[0160] The Applicants concluded that the use of histone-like proteins in the tagging reaction had the following advantages: (i) eliminating the need for accurate dilution of the transposase enzyme; (ii) potentially reducing the observed stability issues of the diluted TnAa enzyme; and (iii) adding some flexibility in DNA concentration.

Claims

1. A composition comprising a histone-like protein and a transposition system, wherein the histone-like protein is an archaeal histone-like protein derived from either the genus Thermococcus or Pyrococcus.

2. The composition according to claim 1, wherein the histone-like protein comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 1 or 2.

3. The composition according to claim 1 or 2, wherein the concentration of the histone-like protein in the composition is in the range of about 2.5 ng / μL to about 25 ng / μL.

4. The composition according to claim 1 or 2, wherein the transposition system comprises a transposase and one or more adapters.

5. The composition according to claim 1 or 2, wherein the transposition system comprises a highly active Tn5 transposase and a Tn5-type transposase recognition site.

6. The composition according to claim 5, wherein the transposition system further comprises one or more oligonucleotides.

7. The composition according to claim 1 or 2, wherein the concentration of the transposition system in the composition is in the range of about 150 ng / μL to about 200 ng / μL.

8. The composition according to claim 1 or 2, further comprising double-stranded DNA, wherein the concentration of the double-stranded DNA in the composition is in the range of about 2 ng / μL to about 8 ng / μL.

9. The composition according to claim 1 or 2, further comprising double-stranded DNA, wherein the ratio of the concentration of the histone-like protein to the concentration of the DNA in the composition is in the range of about 0.5:1 to about 5:

1.

10. Co ２＋ Mn ２＋ Mg ２＋ , Cd ２＋ , and Ca ２＋ The composition according to claim 1 or 2, further comprising a divalent cation selected from the group consisting of the following.

11. The composition according to claim 1 or 2, further comprising a low molecular weight (LMW) buffer containing tris-acetate, glycerol, and DMSO.

12. (a) a first vessel containing histone-like proteins; and (b) A kit comprising a second container including a transposition system; The aforementioned histone-like protein is an archaeal histone-like protein derived from either the genus Thermococcus or Pyrococcus; The transposition system includes a transposase and an adapter; The transposition system comprises: (i) TnAa; or (ii) a highly active Tn5 transposase and a Tn5 type transposase recognition site; A kit comprising the transposition system further comprising one or more oligonucleotides.

13. A method for tagging double-stranded DNA, (a) A step to obtain a sample containing double-stranded DNA; (b) A step of introducing a fragmentation composition containing a histone-like protein and a transposition system into the obtained sample to provide a fragmentation reaction mixture; (c) A step of heating the fragmentation reaction at a predetermined temperature for a predetermined time; and (d) Step of isolating the tagged DNA from the fragmentation reaction mixture. Methods that include...

14. A method for processing a sample containing genomic material, (a) A step of obtaining a tagmentation reaction mixture including a transposition system; (b) A step of introducing double-stranded DNA and histone-like proteins into the tagmentation reaction mixture to provide a fragmentation reaction mixture; and (c) A step of heating the fragmentation reaction mixture to a predetermined temperature for a predetermined time. Methods that include...

15. A method for processing a sample containing genomic material, (a) A step of obtaining a tagmentation reaction mixture comprising a buffer and a transposition system; (b) A step of introducing a solution containing one or more nucleosome-like structures into the tagmentation reaction mixture to provide a fragmentation reaction mixture, wherein the solution containing one or more nucleosome-like structures contains double-stranded DNA that is entangled with or wrapped around one or more histone-like proteins, and (c) A step of heating the fragmentation reaction mixture to a predetermined temperature for a predetermined time. Methods that include...

16. A method for processing a sample containing genomic material, (a) A step of obtaining a sample in a reaction vessel, wherein the sample contains double-stranded DNA material; (b) A step of introducing histone-like proteins into the reaction vessel to provide a DNA histone-like protein solution; (c) A step of introducing the transposition system into the DNA histone-like protein solution to provide a fragmentation reaction; and (d) A step of heating the fragmentation reaction mixture to a predetermined temperature for a predetermined time. Methods that include...

17. A double-stranded DNA fragment having a size in the range of approximately 250 to approximately 300 bp, wherein the double-stranded DNA fragment is (a) A step to obtain a sample containing double-stranded DNA; (b) A step of introducing a fragmentation composition comprising a histone-like protein and a transposition system into the sample to provide a fragmentation reaction; and (c) A step of heating the fragmentation reaction mixture at a predetermined temperature for a predetermined time. A double-stranded DNA fragment prepared by [method / tool ​​name].

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