Control of fragmented and tagged sequencing library insert size using archaea histone-like proteins

Abstract

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

Description

Background Art

[0001] The emergence of single-cell genome amplification technology and next-generation sequencing methods has made breakthroughs in the ability to sequence the genome and transcriptome of individual biological cells. It is now possible to perform massively parallel DNA sequencing on thousands of samples in a single instrument run, but the preparation of individual sequencing libraries is both expensive and time-consuming. Library preparation itself is an essential process before sequencing and includes several aspects that affect sequencing efficiency. Library preparation "usually involves the following major steps: fragmentation of input DNA, end repair and A-tailing of DNA fragments, connection of indexed sequencing adapters, and optional amplification of the connected 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 main bottlenecks of sample preparation is DNA fragmentation. The size of the generated DNA fragments depends on the sequencing platform used and can vary between sequencing technologies that target short reads (e.g. Ion Torrent TM ) to long-read sequencing technologies (e.g., Pacific and Oxford Nanopore )>10kb range. The methods for fragmenting DNA are roughly divided into two basic categories: mechanical and enzyme-based. Mechanical shearing methods include acoustic shearing, hydrodynamic shearing and atomization, while enzyme-based methods include transposons, restriction enzymes and nicking enzymes. Although there are many different options for fragmenting DNA, the final fragment size, the amount of starting material, the initial capital investment and scalability must be considered when selecting a fragmentation method. Crucially, in order to be used for next-generation sequencing, the method used must be sufficiently random to shear the DNA so that the library being sequenced fully represents the original sample.

[0003] Fragmentation and tagging is the process of combining fragmentation and adapter incorporation steps. As used herein, the term "fragmentation and tagging" refers to the combined fragmentation of double-stranded DNA samples catalyzed by transposase and the tagging of fragments with sequences adjacent to transposon end sequences. Hyperactive variants of bacterial Tn5 transposases that mediate the fragmentation of double-stranded DNA and connect synthetic oligonucleotides have been widely used in next-generation sequencing (NGS). Its utility in generating libraries for NGS systems was first described in a paper by Andrew Adey et al. in 2010 (Adey et al., "Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition," Genome Biol 11: R119, 2010, the disclosure of which is hereby incorporated by reference in its entirety). In commercially available products such as Nextera from Illumina and MuSeek from Thermo Scientific, transposases insert NGS system-specific adapter oligonucleotides into double-stranded DNA samples. Such simple one-step fragmentation and tagging reactions have greatly simplified the process of preparing libraries for sequencing, shortening workflow time and reducing costs. Summary of the invention

[0004] The sequencing method using fragmentation and labeling involves the fragmentation of double-stranded DNA and adds universal overhangs at the same time. As mentioned above, due to the combined fragmentation and labeling steps, the workflow allows the rapid generation of sequencing libraries. However, the sequencing library preparation process is highly sensitive to the input DNA concentration and / or the input transposition system concentration. Therefore, it is necessary to accurately quantify the input DNA concentration and / or the transposition system concentration to control the fragment size. The applicant has unexpectedly found that fragmentation and labeling in the presence of one or more histone-like proteins alleviates the need for accurately quantifying the input DNA concentration and / or the transposition system concentration. In addition, the applicant has found that fragmentation and labeling in the presence of one or more histone-like proteins allows the DNA fragment size distribution to be accurately controlled. In addition, the applicant has found that fragmentation and labeling in the presence of one or more histone-like proteins allows better control of the fragment size and labeling fragment size, thereby promoting the greater utility of fragmentation and labeling in applications where longer fragments need to be inserted.

[0005] The 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 archaeon. In some embodiments, the histone-like protein is an archaeal histone-like protein derived from one of the genus Pyrococcus or Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence with 85% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence with 90% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence with 95% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1 to 2. In some embodiments, the concentration of the histone-like protein in the composition is in the range of between about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of the histone-like protein in the composition is in the range of between 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-type transposase recognition site. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase, a Tn5-type transposase recognition site, and one or more oligonucleotides. In some embodiments, the concentration of the transposition system in the composition is in the range of about 150 ng / μL to about 200 ng / μL. In some embodiments, the transposition system concentration range in the composition is about 180 ng / μL.

[0007] In some embodiments, the composition further comprises a nucleic acid sequence, such as DNA, ctDNA, double-stranded DNA, etc. 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 the double-stranded DNA in the composition is in the range of about 2ng / μL to about 8ng / μL. In some embodiments, the concentration of the double-stranded DNA in the composition is in the range of about 3ng / μL to about 7ng / μL. In some embodiments, the concentration of the double-stranded DNA in the composition is about 5ng / μL. In some embodiments, the ratio of the concentration of histone-like proteins to the concentration of DNA in the composition is in the range of about 0.5:1 to about 5:1. In some embodiments, the ratio of the concentration of histone-like proteins to the concentration of DNA in the composition is in the range of about 1:1 to about 4:1. In some embodiments, the ratio of the concentration of histone-like proteins to the concentration of DNA in the composition is in the range of 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 a LMW buffer. In some embodiments, the LMW buffer comprises tris-acetate, glycerol, and DMSO.

[0009] The 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 archaeon. In some embodiments, the histone-like protein is an archaeal histone-like protein derived from one of the genus Pyrococcus or Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence with 85% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence with 90% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence with 95% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1 to 2. In some embodiments, the concentration of the histone-like protein in the composition is in the range of between about 2.5 ng / μL to about 25 ngμL. In some embodiments, the concentration of the histone-like protein in the composition is in the range of between 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 type transposase recognition site. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase, a Tn5 type transposase recognition site and one or more oligonucleotides. In some embodiments, the concentration of the transposition system in the composition is in the range of about 150ng / μL to about 200ng / μL. In some embodiments, the transposition system concentration range in the composition is about 180ng / μL.

[0011] In some embodiments, the concentration of double-stranded DNA in the composition is in the range of about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is in the range of 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 proteins to the concentration of DNA in the composition is in the range of about 0.5:1 to about 5:1. In some embodiments, the ratio of the concentration of histone-like proteins to the concentration of DNA in the composition is in the range of about 1:1 to about 4:1. In some embodiments, the ratio of the concentration of histone-like proteins to the concentration of DNA in the composition is in the range of 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 a LMW buffer. In some embodiments, the LMW buffer comprises tris-acetate, glycerol, and DMSO.

[0013] The third aspect of the present disclosure is a composition comprising a histone-like protein, a transposition system, double-stranded DNA, and divalent cations (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 one of the genus Pyrococcus or Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1 to 2. In some embodiments, the concentration of the histone-like protein in the composition is in the range of between about 2.5 ng / μL to about 25 ngμL. 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 type transposase recognition site. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase, a Tn5 type transposase recognition site and one or more oligonucleotides. In some embodiments, the concentration of the transposition system in the composition is in the range of about 150ng / μL to about 200ng / μL. In some embodiments, the transposition system concentration range in the composition is about 180ng / μL.

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

[0016] The fourth aspect of the present disclosure is a kit comprising: a first container comprising a histone-like protein; and a second container comprising a transposition system. 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 one of the genus Pyrococcus or Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence with 85% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence with 90% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence with 95% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1 to 2. In some embodiments, the concentration of the histone-like protein in the composition is in the range of between about 2.5 ng / μL to about 25 ngμL. In some embodiments, the concentration of the histone-like protein in the first container is in the range of between about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of the histone-like protein in the first container is in a range between about 7.5 ng / μL to about 20 ng / μL.

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

[0018] The fifth aspect of the present disclosure is a method for fragmenting and tagging double-stranded DNA, comprising: (i) obtaining a sample comprising 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; (iii) heating the fragmentation reaction mixture at a predetermined temperature for a predetermined amount of time; and (iv) separating the fragmented and 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 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity with any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence having 95% identity with any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1 to 2. In some embodiments, the concentration of histone-like proteins in the fragmentation reaction mixture is in the range of about 2.5 ng / μL to about 25 ng / μL. In some embodiments, the concentration of histone-like proteins in the fragmentation reaction mixture is in the range of about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of histone-like proteins in the fragmentation reaction mixture is in the range of about 7.5 ng / μL to about 20 ng / μL.

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

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

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

[0022] In some embodiments, the method further comprises introducing a stop solution to the reaction mixture. In some embodiments, the stop solution comprises SDS.

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

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

[0025] In some embodiments, the concentration of double-stranded DNA in the composition is in the range of about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is in the range of 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 proteins in the fragmentation and labeling reaction mixture to the concentration of double-stranded DNA in the fragmentation reaction mixture is in the range of about 1:1 to about 4:1. In some embodiments, the ratio is in the range of about 1.5:1 to about 3:1.

[0026] The 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, the sample comprising 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 sample fragmentation reaction mixture to a predetermined temperature for a predetermined amount of time. In some embodiments, the transposition system comprises a hyperactive mutant of a TnAa or Tn5 transposase and an oligonucleotide material. In some embodiments, the oligonucleotide material comprises a synthetic oligonucleotide. In some embodiments, the histone-like protein is derived from a thermophilic or hyperthermophilic archaea. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence having 90% identity to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence that is 95% identical to 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 is in the range of about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is in the range of 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 proteins in the fragmentation reaction mixture is in the range of about 2.5 ng / μL to about 25 ngμL. In some embodiments, the concentration of histone-like proteins in the fragmentation reaction mixture is in the range of about 5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of histone-like proteins in the fragmentation reaction mixture is in the range of about 7.5 ng / μL to about 20 ng / μL. In some embodiments, the concentration of histone-like proteins in the fragmentation reaction mixture is in the range of about 10 ng / μL to about 20 ng / μL. In some embodiments, the concentration of histone-like proteins in the reaction fragmentation reaction mixture is in the range of about 10 ng / μL to about 15 ng / μL. In some embodiments, the concentration of histone-like proteins in the fragmentation reaction mixture is in the range of about 15 ng / μL to about 20 ng / μL.

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

[0030] In some embodiments, the method further comprises separating the fragmented and tagged DNA from the fragmentation reaction mixture. In some embodiments, the separated fragmented and tagged DNA has a size distribution in the range between about 250bp to about 350bp. In some embodiments, the separated fragmented and tagged DNA has a size distribution in the range between about 250bp to about 320bp. In some embodiments, the separated fragmented and tagged DNA has a size distribution in the range between about 250bp to about 300bp. In some embodiments, the method further comprises amplifying the separated fragmented and tagged DNA to provide a plurality of amplicons. In some embodiments, the method further comprises sequencing the plurality of amplicons. In some embodiments, sequencing the amplicons comprises next generation sequencing.

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

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

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

[0034] In some embodiments, the preparation further comprises removing impurities from the reaction mixture. In some embodiments, removing impurities comprises: (a) capturing the DNA fragments onto beads; (b) washing away impurities from the reaction mixture; and (c) eluting the captured DNA fragments from the beads.

[0035] The 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 NO: 1 to 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 and a solution containing one or more divalent cations.

[0041] The 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 NO: 1 to 2. In some embodiments, the composition further comprises a divalent cation. In some embodiments, the composition further comprises a double-stranded DNA.

[0042] The sixteenth aspect of the present disclosure is a method for processing a sample containing genomic material, comprising: (i) obtaining a fragmentation and labeling reaction mixture containing a transposition system; (ii) introducing double-stranded DNA and histone-like proteins into the fragmentation and labeling reaction mixture to provide a fragmentation reaction mixture; and (iii) heating the fragmentation reaction mixture to a predetermined temperature for a predetermined amount of time. In some embodiments, double-stranded DNA and histone-like proteins are added to the fragmentation and labeling reaction mixture successively. In some embodiments, double-stranded DNA and histone-like proteins are added to the fragmentation and labeling reaction mixture simultaneously. In some embodiments, double-stranded DNA and histone-like proteins are first mixed together to form a DNA-histone-like protein solution, and then the DNA-histone-like protein solution is added to the fragmentation and labeling 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 one of the genus Pyrococcus or Pyrococcus. In some embodiments, the histone-like protein comprises an amino acid sequence having 85% identity with any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence that is 90% identical to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises an amino acid sequence that is 95% identical to any one of SEQ ID NOs: 1 to 2. In some embodiments, the histone-like protein comprises any one of SEQ ID NOs: 1 to 2.

[0043] In some embodiments, the concentration of double-stranded DNA in the composition is in the range of about 2 ng / μL to about 8 ng / μL. In some embodiments, the concentration of double-stranded DNA in the composition is in the range of 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 proteins in the fragmentation reaction mixture to the concentration of double-stranded DNA in the fragmentation and labeling reaction mixture is in the range of about 1:1 to about 4:1. In some embodiments, the ratio is in the range of about 1.5:1 to about 3:1.

[0044] In some embodiments, the transposition system comprises a transposase, a transposon, and an adapter. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase and a Tn5-type transposase recognition site. In some embodiments, the transposition system comprises a hyperactive Tn5 transposase, a Tn5-type transposase recognition site, and one or more oligonucleotides. In some embodiments, the fragmentation and labeling reaction mixture further comprises a divalent cation selected from the group consisting of: Co2+ , Mn 2 + Mg 2+ 、Cd 2+ and Ca 2+ . BRIEF DESCRIPTION OF THE DRAWINGS

[0045] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

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

[0047] Figure 1 Depicted is a method of fragmenting and tagging DNA in the presence of one or more histone-like proteins according to one embodiment of the present disclosure.

[0048] Figure 2 A method of fragmenting and tagging DNA in the presence of one or more histone-like proteins according to one embodiment of the present disclosure is presented.

[0049] Figure 3 A method of fragmenting and tagging DNA in the presence of one or more histone-like proteins according to one embodiment of the present disclosure is presented.

[0050] Figure 4 Depicted are downstream processes that may be performed following fragmentation and tagging of double-stranded DNA.

[0051] Figure 5 Shown are the fragment size distributions of double-stranded DNA after fragmentation and tagging using different concentrations of the transposition system.

[0052] Figure 6 Shown are the fragment size distributions of double-stranded DNA after fragmentation and tagging in the presence of different concentrations of histone-like proteins.

[0053] Figure 7 Shown are insert sizes of fragmented and tagged libraries prepared in the presence of histone-like protein and transposition system, where the concentration of histone-like protein was varied while the concentration of transposition system was kept constant.

[0054] Figure 8 Shown are the insert sizes of fragmented and tagged libraries prepared in the presence of different concentrations of the transposition system. DETAILED DESCRIPTION

[0055] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are presented unless indicated to the contrary.

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

[0057] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one element from a number of elements or a list of elements, but also including more than one element, and optionally including additional unlisted items. Only terms indicating the contrary, such as "only one" or "exactly one", or "consisting of..." used in the claims, will refer to the inclusion of exactly one element from a number of elements or a list of elements. In general, the term "or" as used herein shall be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both") only when it is preceded by an exclusive term such as "or", "one of", "only one", or "exactly one". "Substantially consisting of..." used in the claims shall have the ordinary meaning used in the field of patent law.

[0058] The terms "including", "comprising", "having", etc. are used interchangeably and have the same meaning. Similarly, "including", "comprising", "having", etc. are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "including" under ordinary U.S. patent law, so each term can be understood as an open term, which means "at least the following" and can also be interpreted as not excluding additional features, limitations, aspects, etc. Therefore, for example, "a device having components a, b and c" means that the device includes at least components a, b and c. Similarly, the phrase: "a method involving steps a, b and c" means that the method includes at least steps a, b and c. In addition, although the steps and processes may be outlined in a particular order herein, those skilled in the art will recognize that the order steps and processes may vary.

[0059] As used herein in the specification and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood as at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. In addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, the definition also allows other elements to be optionally present, whether or not they are related to the specifically identified elements. Thus, as a 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") may refer to at least one, optionally including more than one A, without B (and optionally including elements other than B) in one embodiment; to at least one, optionally including more than one B, without A (and optionally including elements other than A) in another embodiment; to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements) in yet another embodiment; and so on.

[0060] References throughout this specification to "one embodiment," "an embodiment," etc., mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in different places in 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 clonal population of polynucleotides that can be single-stranded or double-stranded that is replicated from one or more starting sequences. The one or more starting sequences can be one or more copies of the same sequence, or they can be a mixture of different sequences. In some embodiments, an amplicon is formed by the amplification of a single starting sequence. Amplicon can be produced by various amplification reactions, and the products of these amplification reactions include replicas of the one or more starting or target nucleic acids. On the one hand, the amplification reaction that produces an amplicon is "template-driven", because the base pairing of the reactant (nucleotide or oligonucleotide) has the complement required for forming a reaction product in the template polynucleotide. On the one hand, the template-driven reaction is a primer extension performed with a nucleic acid polymerase or an oligonucleotide connection performed with 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., which are disclosed in the following references (each of which is incorporated herein by reference in its entirety): Mullis et al., U.S. Pat. Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al., U.S. Pat. No. 5,210,015 (real-time PCR with "taqman" probes); Wittwer et al., U.S. Pat. No. 6,174,670; Kacian et al., U.S. Pat. No. 5,399,491 ("NASBA"); Lizardi, U.S. Pat. No. 5,854,033; Aono et al., Japanese Patent Publication JP 4-262799 (rolling circle amplification); etc. In one aspect, the amplicons of the present invention are generated by PCR. The amplification reaction can be a "real-time" amplification that allows measurement of the reaction products as the amplification reaction proceeds (if detection chemistry is available), for example, "real-time PCR" or "real-time NASBA" as described in Leone et al., Nucleic Acids Research, 26:2150-2155 (1998) and similar references.

[0062] As used herein, "amplification" refers to the process of increasing the number of copies. Amplification can be a process in which replication occurs repeatedly over time to form multiple copies of a template. As the amplification proceeds, the amplification can produce an exponential or linear increase in the number of copies. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade RCA, nucleic acid-dependent amplification (NASBA), etc. In addition, amplification can apply linear or circular templates. Amplification can be performed under any suitable temperature conditions, such as thermal cycling or isothermal. In addition, amplification can be performed in an amplification mixture (or reagent mixture), and the amplification mixture (or reagent mixture) is any composition capable of amplifying a nucleic acid target (if any) in a mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve continuous multiple rounds of replication. PCR can be performed by thermal cycling between two or more temperature set points (such as a higher denaturation temperature and a lower annealing / extension temperature) or between three or more temperature set points (such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature, etc.). PCR can be performed using a thermostable polymerase, such as Taq DNA polymerase. PCR typically produces an exponential increase in the amount of product amplicon in successive cycles.

[0063] As used herein, the term "barcode sequence" or "molecular barcode" refers to a unique nucleotide sequence that can be used to: a) identify and / or track the source of a polynucleotide in 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 sequences can vary greatly in size and composition; the following references provide guidance for selecting a set of barcode sequences that are appropriate for a particular embodiment: Casbon (Nuc. Acids Res. 2011, 22e81), Brenner, U.S. Pat. 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. Pat. No. 5,981,179; etc. In particular embodiments, the barcode sequence can have a length ranging from 2 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides.

[0064] As used herein, the terms "biological sample", "tissue sample", "specimen" and the like refer to any sample containing biological molecules (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 humans; mammals such as cats, dogs, horses, cows and pigs; and laboratory animals such as mice, rats and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria and fungi. Biological samples include tissue samples (such as tissue sections and puncture biopsies of tissues), cell samples (such as cytological smears, such as cervical smears or blood smears or cell samples obtained by microdissection) or cell fractions, fragments or organelles (such as obtained by lysing cells and separating their components by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical biopsy or needle biopsy), nipple aspirate, cerumen, breast milk, vaginal secretions, saliva, swabs (e.g., oral swabs), or any material containing biomolecules and derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (e.g., a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0065] As used herein, the term "fragment" refers to a portion of a larger polynucleotide molecule. For example, a polynucleotide can be broken down or fragmented into multiple segments by natural processes (such as the case of cfDNA fragments that can occur naturally within a biological sample) or by in vitro manipulation. The sample can be fragmented via fragmentation and tagging.

[0066] As used herein, the term "mixture" refers to a combination of elements that are dispersed and not in any particular order. A mixture is heterogeneous and cannot be separated into its different compositions in space. Examples of mixtures of elements include multiple different elements dissolved in the same aqueous solution and multiple different elements attached to a solid support at random positions (i.e., not in a particular order). Mixtures are non-addressable. In order to demonstrate by example, as is well known in the art, the array of spatially separated surface-bound polynucleotides is not a mixture of surface-bound polynucleotides, because the types of surface-bound polynucleotides are spatially distinguishable and the array is addressable.

[0067] As used herein, the term "next generation sequencing" refers to a sequencing technology with high-throughput sequencing compared to traditional Sanger-based and capillary electrophoresis-based methods, in which the sequencing process is performed in parallel, such as generating thousands or millions of relatively small sequence reads at a 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 produce shorter reads (from about 25 to about 500 bp), but produce hundreds of thousands or millions of reads in a relatively short period of time. Examples of such sequencing devices available from Illumina (San Diego, CA) include, but are not limited to, iSEQ, MiniSEQ, MiSEQ, NextSEQ, NoveSEQ. It is believed that Illumina next generation sequencing technology uses clonal amplification and synthetic sequencing (SBS) chemistry to achieve rapid sequencing. The process simultaneously identifies DNA bases while incorporating them into nucleic acid chains. Each base emits a unique fluorescent signal when added to the growing chain, which is used to determine the order of the DNA sequence. Non-limiting examples of sequencing devices available from ThermoFisher Scientific (Waltham, MA) include Ion Personal Genome Machine TM (PGM TM) system. It is believed that Ion Torrent sequencing measures the direct release of H+ (proton) when a DNA polymerase binds to a single base. Non-limiting examples of sequencing devices available from Pacific Biosciences (Menlo Park, California) include PacBio Sequel Systems. Non-limiting examples of sequencing devices available from Roche (Pleasanton, CA) are Roche 454. Next-generation sequencing methods can also include nanopore sequencing methods. In general, three nanopore sequencing methods have been used: chain sequencing, in which the bases of DNA are identified as they pass through the nanopore one by one; exonuclease-based nanopore sequencing, in which nucleotides are enzymatically cut from the DNA molecule one by one and monitored as they are captured by the nanopore and pass through the nanopore; and nanopore sequencing by synthesis (SBS) methods, in which identifiable polymer tags are attached to nucleotides and aligned in the nanopore during enzyme-catalyzed DNA synthesis. Common to all these methods is the need to precisely control the reaction rate in order to determine each base in sequence. Chain sequencing requires a method for slowing down the passage of DNA through the nanopore and decoding multiple bases within the channel; for this purpose, a ratchet effect method using molecular motors has been developed. Exonuclease-based sequencing requires the release of each nucleotide close enough to the hole to ensure its capture and its transport through the hole at a sufficiently slow rate to obtain an effective ion current signal. In addition, these methods all rely on the distinction between four natural bases (two relatively similar purines and two similar pyrimidines). The nanopore SBS method utilizes synthetic polymer tags attached to nucleotides, which are specifically designed to produce unique and easily distinguishable ion current blocking features for sequence determination. In some embodiments, nucleic acid sequencing via nanopore sequencing includes: preparing a nanopore sequencing complex and determining the polynucleotide sequence. Methods for preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017 / 0268052 and 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, labeled nucleotides can be used to determine polynucleotide sequences (see, e.g., PCT Publications WO / 2020 / 131759, WO / 2013 / 191793, and WO / 2015 / 148402, the disclosures of which are hereby incorporated by reference in their entirety). Analysis of the data generated by sequencing is typically performed using software and / or statistical algorithms that perform various data conversions, e.g., converting signal emissions into base calls, converting base calls into consensus sequences of nucleic acid templates, etc.Such software, statistical algorithms, and their use are described in detail in U.S. Patent Application Publication Nos. 2009 / 00243312017 / 0044606 and PCT Publication No. WO / 2018 / 034745, the disclosures of which are hereby incorporated by reference in their entirety.

[0068] As used herein, the term "oligonucleotide" refers to a nucleotide single-stranded polymer having a length of from about 2 to 200 nucleotides, up to 500 nucleotides. Oligonucleotides can be synthetic or can be prepared enzymatically, and in some embodiments have a length of 30 to 150 nucleotides. Oligonucleotides can contain ribonucleotide monomers (i.e., can be oligoribonucleotides), or deoxyribonucleotide monomers, or both ribonucleotide monomers and deoxyribonucleotide monomers. For example, the length of an oligonucleotide can be 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.

[0069] As used herein, the term "sequence" when used to refer to a nucleic acid molecule refers to the order of nucleotides (or bases) in a nucleic acid molecule. In the case of different types of nucleotides in a nucleic acid molecule, the sequence includes the identification of the types of nucleotides (or bases) at various positions in the nucleic acid molecule. A sequence is a characteristic of all or part of a nucleic acid molecule. The term can be similarly used to describe the order and positional identity of monomeric units in other polymers (e.g., amino acid monomeric units of protein polymers).

[0070] As used herein, the term "sequencing" refers to determining the order and position of bases in a nucleic acid molecule. More specifically, the term "sequencing" refers to a biochemical method for determining the order of nucleotide bases, adenine, guanine, cytosine, and thymine in a DNA oligonucleotide. As used herein, sequencing may include, but is not limited to, parallel sequencing or any other sequencing method known to those skilled in the art, such as chain termination, rapid DNA sequencing, wandering-spot analysis, Maxam-Gilbert sequencing, dye terminator sequencing, or use any other modern automated DNA sequencing instrument.

[0071] As used herein, the term "fragmentation and tagging" or "tagmenting and fragmentation" refers to a process in which genomic DNA is cut, tagged with an adapter sequence, and extended to fill the gaps caused by the cutting and tagging. More specifically, "fragmentation and tagging" refers to the modification of DNA by a transposome complex comprising a transposase complexed with an adapter comprising a transposon end sequence. Fragmentation and tagging causes simultaneous fragmentation of DNA and connection of the adapter to the 5' ends of two duplex fragment chains. Fragmentation and tagging causes simultaneous fragmentation of nucleic acids and connection of the adapter to the 5' ends of two duplex fragment chains. After a purification step to remove the transposase, additional sequences may be added to the ends of the appropriate fragments, for example, by PCR, ligation, or any other suitable method known to those skilled in the art.

[0072] As used herein, the phrase "transposition reaction" refers to a reaction in which one or more transposons are inserted into a random site or nearly random site in a target nucleic acid. The essential components in the transposition reaction are a transposase and a DNA oligonucleotide that exhibits a transposon nucleotide sequence, including a transferred transposon sequence and its complement (i.e., a non-transferred transposon end sequence), and other components required to form a functional transposon or transposome complex. As needed or desired, the DNA oligonucleotide may further comprise additional sequences (e.g., an adapter or primer sequence).

[0073] Overview

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

[0075] Composition

[0076] In one aspect of the disclosure are compositions for use in fragmentation and tagging reactions. In some embodiments, the composition comprises 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, for example, divalent cations, buffers, polyols, DMSO, etc.

[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, e.g., divalent cations, buffers, polyols, DMSO, etc.

[0079] In some embodiments, the present disclosure also provides a fragmentation and tagging 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] Each of the components of a fragmentation composition, a fragmentation reaction mixture, and a fragmentation and tagging reaction mixture are described herein.

[0082] Histone-like proteins

[0083] As described above, the disclosed fragmentation compositions and fragmentation reaction mixtures each contain one or more histone-like proteins. Histone-like proteins (HLPs) are small, alkaline bacterial proteins that are associated with the nucleoid and play a role in maintaining DNA architecture and regulating DNA events such as replication, recombination / repair, and transcription. Architectural chromatin proteins are present in every domain of life. In eukaryotes and most archaeal lineages, histones are responsible for packaging and compaction of DNA. Archaeal histone-like proteins show some homology to eukaryotic core histones in primary sequence, secondary, and tertiary structure.

[0084] It is believed that histone-like proteins form homologous and / or heterologous dimers, and play a role in wrapping DNA by forming a tetramer composed of dimers in dimers to form a so-called 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 compress DNA (for HphA homotetramers) by forming these nucleosome-like structures that wrap DNA around proteins with footprints of about 90bp. When these histone-like proteins form nucleosome-like structures, it is believed that they provide a certain degree of nuclease protection by sterically hindering or blocking the recognition site of nuclease to nuclease.

[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 Pyrococcus. 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 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 Thaumarchaeota, Eosarchaeota, Crenarchaeota, Archaeota (TACK), Prohaloarchaeota, Pacearchaeota, Mysteryarchaeota, Nanoarchaeota, Nanohaloarchaeota (DPANN), and Asgard Archaea. Still further archaeal histone-like proteins may be derived from Asgard Archaea and candidate phyla Deep Archaeota, Ussarchaeota, Pacearchaeota, Mysteryarchaeota, Prohaloarchaeota, Huberarchaea, and Micrarchaeota. Even further archaeal histone-like proteins are described by 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 hereby incorporated 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 contiguous nucleotide sequence is SEQ ID NO: 4.

[0090] In some embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 1.5 ng / μL to about 50 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 2 ng / μL to about 40 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 2 ng / μL to about 35 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 2.5 ng / μL to about 35 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 2.5 ng / μL to about 30 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 2.5 ng / μL to about 25 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 2.5 ng / μL to about 20 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 3ng / μL to about 30ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 3ng / μL to about 25ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 3ng / μL to about 20ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 4ng / μL to about 35ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 4ng / μL to about 30ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 4ng / μL to about 25ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 4ng / μL to about 20ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 5 ng / μL to about 35 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 5 ng / μL to about 30 ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 5 ng / μL to about 25 ng / μL.In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 5ng / μL to about 15ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 6ng / μL to about 30ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 6ng / μL to about 25ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 6ng / μL to about 20ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 6ng / μL to about 15ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 7ng / μL to about 30ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of about 7ng / μL to about 25ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of between about 7ng / μL to about 20ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of between about 7ng / μL to about 15ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of between about 8ng / μL to about 25ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of between about 8ng / μL to about 20ng / μL. In other embodiments, the concentration of histone-like proteins in any composition or reaction mixture is in the range of between about 8ng / μL to about 15ng / 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 is at least 85% identical to any one of SEQ ID NOs: 1 and 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 is at least 85% identical to any one of SEQ ID NOs: 1 and 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 is at least 85% identical to any one of SEQ ID NOs: 1 and 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 is at least 85% identical to any one of SEQ ID NOs: 1 and 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 to any one of SEQ ID NOs: 1 and 2, and wherein the concentration of the histone-like protein in any composition or reaction mixture is about 20 ng / μL.

[0093] dsDNA

[0094] In some embodiments, the compositions and / or reaction mixtures disclosed herein include double-stranded DNA. In some embodiments, the double-stranded DNA is cDNA, ctDNA or cfDNA. Of course, it will be appreciated by those skilled in the art that any DNA suitable for use in the present disclosure can be first 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 including one or more organisms. The sources of nucleic acid molecules include, but are not limited to, organelles, cells, tissues, organs, organisms, single cells, or single organelles. Cells useful as a source of target nucleic acid molecules can be 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; or eukaryotic, 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 (e.g., a human patient). In some embodiments, the double-stranded DNA is derived from a tumor sample, e.g., from a tumor sample derived from a human patient.

[0097] In some embodiments, double-stranded DNA can be enriched for certain sequences of interest prior to fragmentation and tagging. U.S. Patent Nos. 10,590,471, 10,900,068, 10,907,204, and 9,365,897; U.S. Patent Publication Nos. 2020 / 0048694 and 2020 / 0392483; and PCT Publication WO / 2012 / 108864 describe various methods for enriching for sequences of interest, the disclosures of which are hereby incorporated by reference herein in their entirety.

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

[0100] Transposition system

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

[0102] In some embodiments, the transposition system comprises a transposase, a transposon or a transposon DNA and one or more oligonucleotides (e.g., a barcode, a tag, an adapter, etc.). In some embodiments, the transposase is compounded 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, wherein each end is 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 when in contact, thereby forming a transposase / transposon DNA complex dimer referred to as a transposome. In some embodiments, the transposome has the ability to bind to the target position along the double-stranded nucleic acid, thereby forming a complex comprising a transposome and double-stranded genomic DNA. Such transposition systems are described in U.S. Pat. No. 10,894,980, U.S. Pat. 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 compounded with the transposon DNA comprises a dimer of a transposase and a pair of adapters (see U.S. Patent Publication No. 2018 / 0305683, the disclosure of which is hereby incorporated by reference in its entirety herein). The term "adaptor" refers to a nucleic acid that can be connected to at least one chain of a double-stranded nucleic acid molecule (e.g., double-stranded DNA) via a transposase-mediated reaction. The adapter can be at least partially double-stranded, and the length can be 30 to 150 bases, for example, 40 to 120 bases. In other embodiments, the transposase complex comprises a transposase loaded with two adapter molecules, each of which comprises a recognition sequence for a transposase at one end. In yet other embodiments, the transposase compounded with the transposon DNA comprises a dimer of a pair of Tn-5 binding double-stranded DNA oligonucleotides containing 19 base pairs of transposase binding sequences (mosaic ends) or inverted repeats. In even further embodiments, the transposition system comprises: at least one first oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one first recognition end sequence; at least one second oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one second recognition end sequence; and a transposase (see PCT Publication No. WO / 2015 / 089339, the disclosure of which is hereby incorporated by reference herein in its entirety).

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

[0105] In some embodiments, the transposition system comprises TnAa. In some embodiments, the transposition system can utilize a hyperactive Tn5 transposase and a Tn5-type transposase recognition site (Goryshin and Reznikoff, J. Biol. Chem., 273:7367 (1998)), or a MuA transposase and a Mu transposase recognition site comprising R1 and R2 terminal 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 & Boeke, Nucleic Acids Res., 22:3765-72, 1994 and International Publication WO 95 / 23875), transposon Tn7 (Craig, NL, Science. 271:1512, 1996; Craig, NL, 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 DJ, et al., EMBO J., 15:5470-9, 1996), Tc1 (Plasterk RH, Curr. Topics Microbiol. Immunol., 204:125-43, 1996), P factor (Gloor, GB, Methods Mol. Biol., 260:97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265:18829-32, 1990), bacterial insertion sequences (Ohtsubo & 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 & Corces, Annu Rev Microbiol. 43:403-34, 1989).Still further transposition systems may include ISS, Tn10, Tn903, IS911, and engineered versions of the transposase family enzymes (Zhang et al., (2009) PLoS Genet. 5:el000689. Epub 2009 Oct. 16; Wilson C. et al. (2007) J. Microbiol. Methods 71:332-5).

[0106] Even further useful transposition systems include the Tn3 transposon 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 transposition system (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, PB, Jr., et al. (2013), PiggyBac transposase tools for genome engineering, Proc. Natl. Acad. Sci. USA 110, E2279-2287), Sleeping Beauty transposition system (see Ivies, Z., Hackett, PB, Piasterk, RH, 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), Tol2 transposition system (see Kawakami, K. (2007), Tol2: a versatile gene transfer vector in vertebrates, Genome Biol. 8 Suppl. 1, S7.). Yet other suitable transposition systems include those provided by Illumina, for example, in the NEXTERA DNA or NEXTERA DNA Flex library preparation kits. Yet even further transposition systems, or components thereof, are described in U.S. Patent Nos. 7,608,434, 7,083,980, 5,965,443, and 5,925,545, the disclosures of which are hereby incorporated by reference in their entireties.

[0107] In some embodiments, the transposase contained within 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 is in the range of about 100ng / μL to about 400ng / μL. In some embodiments, the concentration of the transposition system in any composition or reaction mixture is in the range of about 100ng / μL to about 250ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is in the range of about 120ng / μL to about 230ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is in the range of about 140ng / μL to about 210ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is in the range of about 150ng / μL to about 200ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is in the range of about 160ng / μL to about 190ng / μL. In other embodiments, the concentration of the transposition system in any composition or reaction mixture is about 160ng / 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 cations

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

[0111] In some embodiments, any of the compositions or reaction mixtures of the present disclosure may comprise a concentration of divalent cations 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, any of the compositions or reaction mixtures of the present disclosure may have 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 CoCl 2 In some embodiments, any of the compositions or reaction mixtures of the present disclosure may have a MnCl 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. 2 In some embodiments, any of the compositions or reaction mixtures of the present disclosure may have a MgCl 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. 2 In some embodiments, any of the compositions or reaction mixtures of the present disclosure may have 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 CdCl 2 In some embodiments, any of the compositions or reaction mixtures of the present disclosure may have a CaCl 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. 2concentration.

[0112] Buffer

[0113] In some embodiments, any one of the compositions or reaction mixtures of the present disclosure comprises one or more buffers. Non-limiting examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethyl barbituric acid, piperazine-N, N'-bis (2-ethane sulfonic acid) (PIPES), dimethyl arsenic acid, 2- (N-morpholino) ethane sulfonic acid, tris (hydroxymethyl) methylamine (TRIS), 2- (N-morpholino) ethane sulfonic acid (TAPS), N, N-bis (2-hydroxyethyl) glycine (Bicine), N-tris (hydroxymethyl) methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethane sulfonic acid (HEPES), 2-{[tris (hydroxymethyl) methyl] amino} ethane sulfonic acid (TES) and combinations thereof. In other embodiments, the buffer solution can be composed 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 a combination thereof.

[0114] Additional components

[0115] In some embodiments, any one of the compositions or reaction mixtures of the present disclosure comprises a polyol. Suitable polyols include 1,2-ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 2-methyl-1,3-propylene glycol, 2-methyl-1,2-propylene glycol, 2,2-dimethyl-1,3-propylene glycol, 2,2-diethyl-1,3-propylene glycol, 2-methyl-2-propyl-1,3-propylene glycol, 2-butyl-2-ethyl-1,3-propylene glycol, dihydroxyacetone, 2,2-dibutyl-1,3-propylene glycol, 3-methoxy-1,3-propylene glycol, 3-methoxy-1,2-propylene glycol, 3-methoxy-2,3-propylene glycol, 2-methoxymethyl-1,3-propylene glycol, 3-ethoxy-1,3-propylene glycol, 3-ethoxy-1,2-propylene glycol, 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, ,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-nonanediol, 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 diol, 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-cycloheptanediol, 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-Cycloheptantriol, 1,2,4-Cycloheptantriol, 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 triol, 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-octantriol, 1,2,8-octantriol, 1,2,3-nonanetriol, 1,2,9-nonanetriol, 1,2,3- Decanetriol, 1,2,10-decanetriol, 1,2,3-undecantriol, 1,2,11-undecantriol, 1,2,3-dodecanetriol, 1,1,12-dodecanetriol, 2,2-bis(hydroxymethyl)-1,3-propanediol, 1,2,3,4-butanetetrol, 1,2,3,4-pentanetetrol, 1,2,3,5-pentanetetrol, 1,2,3,4-hexanetetrol, 1,2,3,6-hexanetetrol, 1,2,3,4-heptanetetrol Tetraol, 1,2,3,7-heptetrol, 1,2,3,4-octanetrol, 1,2,3,8-octanetrol, 1,2,3,4-nonanetetrol, 1,2,3,9-nonanetetrol, 1,2,3,4-decanetrol, 1,2,3,10-decanetrol, trimethylolpropanol, pentaerythritol, sugar alcohols such as mannitol, sorbitol or arabitol, hexadecene, 1,2,3,4,5-pentapentol and 1,2,3,4,5,6-hexadecene. In some embodiments, the polyol is 1,2,3-propanetriol (also known as glycerol).

[0116] In some embodiments, any of the compositions or reaction mixtures of the present disclosure comprises dimethyl sulfoxide.

[0117] In some embodiments, any of the compositions or reaction mixtures of the present disclosure include one or more salts or surfactants.

[0118] Fragmentation and labeling methods in the presence of histone-like proteins

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

[0120] refer to Figure 1, first obtain a sample (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 (e.g., 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 (step 102) to the obtained sample to provide a fragmentation reaction mixture. As described herein, the fragmentation composition may include one or more histone-like proteins, one or more transposition systems, and one or more optional additional components. In some embodiments, the sample obtained is allowed to mix with the fragmentation composition for a period of time before heating. In some embodiments, the sample obtained is allowed to mix 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 heating.

[0122] Subsequently, the fragmentation reaction mixture is heated at a predetermined temperature for a predetermined amount of time (step 103), i.e., the fragmentation and tagging reaction is performed by heating the fragmentation reaction mixture. In some embodiments, the fragmentation and tagging reaction can be performed 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 fragmentation and tagging reaction can be performed 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 fragmentation and tagging reaction can be performed for a period of time ranging from 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 fragmentation and tagging reaction can be performed for about 2 minutes; for about 3 minutes; for about 4 minutes; for about 5 minutes; or for about 6 minutes. Methods and additional reaction conditions for fragmenting and tagging DNA using a transposition system are described in U.S. Pat. No. 9,080,211 and U.S. Pat. Pub. No. US2010 / 0120098, the disclosures of which are hereby incorporated by reference herein in their entirety.

[0123] Next, the fragmented and tagged DNA is separated from the fragmentation reaction mixture (step 104). In some embodiments, the separation of the fragmented and tagged DNA includes: (a) capturing the generated DNA fragments onto beads using capture probes; (b) washing away impurities and non-targeted fragments from the reaction mixture; and (c) eluting or releasing the captured DNA fragments from the beads or capture probes.

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

[0125] Figure 2 An alternative method for fragmenting and tagging DNA is shown in . In this embodiment, a fragmentation and tagging reaction mixture is first obtained (step 201). Subsequently, double-stranded DNA and histone-like proteins are introduced into the fragmentation and tagging reaction mixture to form a fragmentation reaction mixture (step 202). In some embodiments, 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 the histone-like protein are added simultaneously. In yet other embodiments, the double-stranded DNA and the histone-like protein are first combined to form a DNA-histone-like protein solution, which is then added to the fragmentation and labeling reaction mixture to form a fragmentation reaction mixture. Next, the fragmentation reaction mixture is heated at a predetermined temperature for a predetermined amount of time (step 103). After fragmentation and labeling, the fragmented and tagged DNA is separated (step 104). In some embodiments, the fragmented and tagged DNA is "cleaned up" to remove impurities before amplification. In some embodiments, "cleaning" utilizes functionalized beads, such as KAPA PureBeads (KAPA Biosystems, Inc., Wilmington, Massachusetts).

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

[0127] Kit Components

[0128] The present disclosure also provides a test kit comprising any one of the compositions or reaction mixtures described herein. In some embodiments, the test kit disclosed herein may optionally contain other components, including but not limited to reagents (e.g., PCR primers, polymerase, buffers, nucleotides, etc.) for performing polymerase chain reaction. As required, the various components of the test kit of the present disclosure may be present in independent containers, or some compatible components may be pre-merged into a single container.

[0129] In some embodiments, any one of the kits disclosed herein may further include one or reagents for performing polymerase chain reaction (PCR). In some embodiments, PCR reagents include deoxynucleoside triphosphates (dNTPs), particularly all four naturally occurring deoxynucleoside triphosphates (dNTPs). In some embodiments, PCR reagents include deoxyribonucleoside triphosphate molecules, including all of dATP, dCTP, dGTP, and dTTP. In some embodiments, PCR reagents also include compounds that can be used to assist nucleic acid polymerase activity. For example, in some embodiments, PCR reagents include divalent cations, such as magnesium ions. In some embodiments, magnesium ions are provided in the form of magnesium chloride, magnesium acetate, or magnesium sulfate. In some embodiments, PCR reagents further include buffers or buffer solutions, including any one of the buffers described herein.

[0130] In some embodiments, any of the kits disclosed herein may further include at least one polymerase, a modified polymerase, or a thermostable polymerase. As used herein, the term "polymerase" refers to an enzyme that performs template-guided synthesis of polynucleotides. DNA polymerase can only add free nucleotides at the 3' end of the newly formed chain. This causes the newly formed chain to elongate in the 5'-3' direction. No known DNA polymerase can start a new chain (from the beginning). DNA polymerase can only add nucleotides on a pre-existing 3'-OH group, so a primer is required where it 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, telomerases, reverse transcriptases, and RNA polymerases. Reverse transcriptases are RNA-dependent DNA polymerases that can synthesize DNA from RNA templates. The reverse transcriptase family contains DNA polymerase function and RNase H function, which can degrade paired RNA bases into DNA. RNA polymerases are enzymes that use DNA as a template to synthesize RNA during gene transcription. RNA polymerase polymerizes ribonucleotides at the 3' end of the RNA transcript.

[0131] In some embodiments, suitable polymerases may be derived from: Archaea (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 strain KOD (Pfx, GenBank: AAE68738), Thermococcus caucasus (Thermococcus gorgonarius)(Tgo,Pdb:4699806), Sulfolobus solataricus(GenBank:NC002754,P26811), Aeropyrumpernix(GenBank:BAA81109), Archaeglobus fulgidus(GenBank:029753), Pyrobaculum aerophilum(GenBank:AAL63952), Pyrodictium occultum(GenBank:BAA07579,BAA07580), Thermococcus 9 degrees Nm(GenBank:AAA88769,Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus GE8 (GenBank: CAC12850), Thermococcus JDF-3 (GenBank: AX135456, WO0132887), Thermococcus TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (GenBank: NP143776), Pyrococcus GE23 (GenBank: CAA90887), Pyrococcus ST700 (GenBank: CAC 12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus xikuer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus xikuer (GenBank: DD259850.1), Thermococcus xikuer (GenBank: DD259850.1), Thermococcus xikuer (GenBank: DD259850.1), Thermococcus xikuer (GenBank: DD259850.1), Thermococcus xikuer (GenBank: E14137 ... siculi)(GenBank:DD259857.1), Thermococcusthioreducens, Thermococcus onnurineusNA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum(GenBank:AAF27815), Methanococcus jannaschii jannaschii (GenBank: Q58295), Desulfurococcus species TOK, Desulfurococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025, and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579), thermophilic bacteria Thermus species (e.g., Thermus flavus, Thermus ruber, Thermus thermophilus, Thermus lacteus, Thermus rubens, Thermus aquaticus, Thermus rub ... aquaticus) and Bacillus stearothermophilusstearothermophilus), Thermotogamaritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, Thermococcus sp. GT, Pyrococcus sp. GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis, and Thermococcus sp. 9N-7 polymerase.

[0132] As used herein, the term "modified DNA polymerase" refers to a DNA polymerase derived from another (i.e., parent) DNA polymerase, and contains one or more amino acid changes (e.g., amino acid substitutions, deletions, or insertions) compared to the parent DNA polymerase. In some embodiments, the modified DNA polymerase of the present disclosure is derived from or modified from a naturally occurring or wild-type DNA polymerase. In some embodiments, the modified DNA polymerase of the present disclosure is derived from or modified from a recombinant or engineered DNA polymerase, including but not limited to a chimeric DNA polymerase, a fusion DNA polymerase, or another modified DNA polymerase. Typically, the modified DNA polymerase has at least one altered phenotype compared to the parent 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 heat-stable, which is heat-resistant and retains sufficient activity to achieve subsequent polynucleotide extension reactions after the time required for achieving double-stranded nucleic acid denaturation at high temperatures, and will not be irreversibly denatured (inactivated). The necessary heating conditions for nucleic acid denaturation are well known in the art and are illustrated in, for example, U.S. Patent Nos. 4,683,202, 4,683,195 and 4,965,188, which are incorporated herein by reference. As used herein, thermostable polymerases are suitable for use in temperature cycling reactions such as polymerase chain reaction ("PCR"), primer extension reactions or end modification (e.g., terminal transferase, degradation or error correction (polishing)) reactions. Irreversible denaturation for the purposes of this article refers to permanent and complete loss of enzyme activity. For thermostable polymerases, enzyme activity refers to the catalytic action of combining nucleotides in an appropriate manner to form polynucleotide extension products complementary to the template nucleic acid chain. Thermostable DNA polymerases from thermophilic bacteria include, for example, DNA polymerases from Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermus species sps17, Thermus species Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, Thermosiphoafricanus, and other thermostable DNA polymerases disclosed above.

[0134] In some embodiments, the polymerase can be a modified naturally occurring A-type polymerase. Further embodiments of the invention generally relate to a method wherein the modified A-type polymerase can be selected from any of the following genera: Meiothermus, Thermotoga, or Thermomicrobium, for example in primer extension, end modification (e.g., terminal transferase, degradation, or error correction), or amplification reactions. Another embodiment of the invention generally pertains to a method wherein the polymerase can be isolated from any of the following: Thermus aquaticus (Taq), Thermus thermophilus, Thermus caldophilus, or Thermus filiformis, for example in primer extension, end modification (e.g., terminal transferase, degradation, or error correction), or amplification reactions. Further embodiments of the invention generally encompass a method wherein a modified A-type polymerase can be isolated from Bacillus stearothermophilus, Sphaerobacter thermophilus, Dictoglomus thermophilum, or Escherichia coli, e.g., in a primer extension, end modification (e.g., terminal transferase, degradation, or error correction), or amplification reaction. In another embodiment, the invention generally relates to a method wherein the modified A-type polymerase can be a mutant Taq-E507K polymerase, e.g., in a primer extension, end modification (e.g., terminal transferase, degradation, or error correction), or amplification reaction. Another embodiment of the invention generally pertains to a method wherein a thermostable polymerase can be used to cause amplification of 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 Therphage Ligase or a derivative thereof, such as Circligase. TM Circligase TM II (Epicentre Tech., Madison, Wisconsin). In other embodiments, the ligase is T4 ligase.

[0136] In addition to the above-mentioned components, the kit of the present invention may further include instructions for practicing the inventive method using the components of the kit, i.e., instructions for library preparation. The instructions for practicing the inventive method are usually recorded on a suitable recording medium. For example, the instructions can be printed on a substrate (such as paper or plastic, etc.). Therefore, the instructions can be present in the kit as a package insert, in the labeling of the container of the kit or its components (i.e., associated with packaging or sub-packaging). In other embodiments, the instructions exist as an electronic storage data file present in a suitable computer-readable storage medium (e.g., CD-ROM, floppy disk, etc.). In other embodiments, the actual instructions are not present in the kit, but are provided for (e.g., via the Internet) to obtain the means for instructions from a remote source. The example of this embodiment is a kit, which includes a network address where the instructions can be viewed and / or the instructions can be downloaded therefrom.

[0137] In some embodiments, any of the kits disclosed herein may include a sequencing device, such as a sequencing device for "next generation sequencing". As used herein, the term "next generation sequencing" refers to a sequencing technology with high-throughput sequencing compared to traditional Sanger-based and capillary electrophoresis methods, in which the sequencing process is performed in parallel, such as generating thousands or millions of relatively small sequence reads at a 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 produce shorter reads (from about 25 to about 500 bp), but generate hundreds of thousands or millions of reads in a relatively short period of time.

[0138] Examples of such sequencing devices available from Illumina (San Diego, CA) include, but are not limited to, iSEQ, MiniSEQ, MiSEQ, NextSEQ, NoveSEQ. It is believed that Illumina next-generation sequencing technology uses clonal amplification and synthetic sequencing (SBS) chemistry to achieve rapid sequencing. The process simultaneously identifies DNA bases while incorporating them into nucleic acid chains. Each base emits a unique fluorescent signal when added to the growing chain, which is used to determine the order of the DNA sequence.

[0139] Non-limiting examples of sequencing devices available from ThermoFisher Scientific (Waltham, MA) include the Ion Personal Genome Machine TM (PGM TM) system. It is believed that Ion Torrent sequencing measures the direct release of H+ (proton) when DNA polymerase binds a single base. Non-limiting examples of sequencing devices available from Pacific Biosciences (Menlo Park, CA) include PacBio Sequel Systems. Non-limiting examples of sequencing devices available from Roche (Pleasanton, CA) are Roche 454.

[0140] Next generation sequencing methods may also include nanopore sequencing methods. In general, three nanopore sequencing methods have been used: chain sequencing, in which the bases of DNA are identified as they pass through the nanopore one by one; exonuclease-based nanopore sequencing, in which nucleotides are enzymatically cut from the DNA molecule one by one and monitored as they are captured by the nanopore and pass through the nanopore; and nanopore sequencing by synthesis (SBS) methods, in which identifiable polymer tags are attached to nucleotides and aligned in the nanopore during enzyme-catalyzed DNA synthesis. What all these methods have in common is the need to precisely control the reaction rate in order to determine each base in sequence.

[0141] Chain sequencing requires a method for slowing down the passage of DNA through the nanopore and decoding multiple bases within the channel; for this purpose, a ratchet effect method using molecular motors has been developed. Exonuclease-based sequencing requires the release of each nucleotide close enough to the hole to ensure its capture and its transport through the hole at a sufficiently slow rate to obtain an effective ion current signal. In addition, these methods all rely on the distinction between four natural bases (two relatively similar purines and two similar pyrimidines). The nanopore SBS method utilizes synthetic polymer tags attached to nucleotides, which are specifically designed to produce unique and easily distinguishable ion current blocking features for sequence determination. In some embodiments, nucleic acid sequencing via nanopore sequencing includes: preparing a nanopore sequencing complex and determining the polynucleotide sequence. Methods for preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017 / 0268052 and PCT Publications WO2014 / 074727, WO2006 / 028508, WO2012 / 083249, and WO / 2014 / 074727, the disclosures of which are hereby incorporated by reference in their entireties. In some embodiments, labeled nucleotides can be used to determine polynucleotide sequences (see, e.g., PCT Publications WO / 2020 / 131759, WO / 2013 / 191793, and WO / 2015 / 148402, the disclosures of which are hereby incorporated by reference in their entireties).

[0142] In some embodiments, any kit of the present disclosure may include software for analyzing the sequencing data obtained. Analysis of the data generated by sequencing is usually performed using software and / or statistical algorithms that perform various data conversions, for example, converting signal emission to base calls, converting base calls to consensus sequences of nucleic acid templates, etc. Such software, statistical algorithms and their use 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 herein by reference in their entirety.

[0143] Examples

[0144] Fragmentation and tagging of DNA in the presence of histone-like proteins was demonstrated using E. coli DNA 10 ng (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 proteins without using dilution of the transposition system concentration to control fragment size. Fragmentation and tagging reaction settings containing between 2.5 ng and 80 ng of total input of the transposition system (TnAa) produced similar library size ranges compared to fragmentation and tagging in the presence of histone-like proteins (compare Figure 5 and Figure 6 ).

[0145] The components for the reaction setup are provided below.

[0146] E. coli DNA 10 ng (mg1655) was fragmented and tagged, and the following TnAa (Tn5L1) enzyme concentrations were used to prepare sequencing libraries: 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.

[0147] Fragmentation and labeling reaction mixture

[0148]

[0149] LMW Buffer (2X):

[0150]

[0151] Dilution buffer

[0152]

[0153] Storage buffer

[0154] <![CDATA[ Components ]]> <![CDATA[ concentration ]]> Formulation Buffer 50％ PIPES, pH 7.5 20 glycerin 26.25

[0155] In the presence of histone-like protein (HphA), 10 ng (mg1655) of E. coli DNA was fragmented and tagged. The fragmentation reaction mixture contained: a transposition system at a concentration of 180 ng / μL; and histone-like protein in an amount 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. The DNA concentration was kept constant at 5 ng / μL in each experiment.

[0156] Fragmentation reaction mixture

[0157]

[0158]

[0159] The fragmentation and tagmentation reaction conditions were as follows:

[0160] 52℃ for 5min

[0161] Add 5 μL of stop solution (0.25% SDS) [What else does the stop solution contain?]

[0162] 0.8x Kapa Pure Bead Purification

[0163] Elute with 22 μL 10 mM Tris

[0164] Amplification was performed after preparation of fragmented and tagged DNA using the following parameters:

[0165]

[0166] temperature time cycle 72℃ 3min 1 98℃ 30sec 98℃ 10sec 12 63℃ 30sec 72℃ 3min 1

[0167] The samples were then sequenced on an Ilumina MiniSeq using the MiniSeq System Mid-Output Kit (2×150 bp). Sequencing reads were quality filtered, sampled, and evaluated based on insert size and insert start site sequence deviation.

[0168] The average insert size of the fragmented and tagged library increased with increasing amounts of histone-like protein added (see Figure 7 When 20 ng to 40 ng of histone-like protein was added, the insert size was stabilized at an insert size of 300 bp (see Figure 7 The start site bias was not affected by the amount of histones added to the fragmentation and tagging reaction (data not shown). The average insert size of the library decreased with increasing amounts of TnAa enzyme (see Figure 8 ). The results showed that larger insert sizes could be obtained using stock concentrations (180 ng / μL) of the transposase, with average insert sizes up to 300 bp.

[0169] Applicants conclude that using histone-like proteins in fragmentation and tagging reactions has the following advantages: (i) eliminates the need for precise transposase dilution; (ii) potentially reduces observed stability issues with diluted TnAa enzyme; and (iii) potentially increases 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 one of the genus Thermococcus or Pyrococcus.

2. The composition of claim 1, wherein the histone-like protein comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 1 or 2.

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

4. The composition of claims 1 to 3, wherein the transposition system comprises a transposase and one or more adapters.

5. The composition according to claims 1 to 4, wherein the transposition system comprises a hyperactive Tn5 transposase and a Tn5-type transposase recognition site.

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

7. The composition of claims 1 to 6, wherein the concentration of the transposition system in the composition is in the range of between about 150 ng / μL to about 200 ng / μL.

8. The composition of claims 1 to 7, further comprising double-stranded DNA, wherein the concentration of the double-stranded DNA in the composition ranges from about 2 ng / μL to about 8 ng / μL.

9. The composition of claims 1 to 7, further comprising double-stranded DNA, wherein the ratio of the concentration of the histone-like protein to the concentration of DNA in the composition is in the range of between about 0.5:1 to about 5:

1.

10. The composition according to claims 1 to 9, further comprising a divalent cation selected from the group consisting of: Co 2+ , Mn 2+ Mg 2+ 、Cd 2+ and Ca 2+ .

11. The composition of claims 1 to 10, further comprising a low molecular weight (LMW) buffer comprising tris-acetate, glycerol and DMSO.

12. A kit comprising: (a) a first container comprising a histone-like protein; as well as (b) a second container comprising a transposition system; wherein the histone-like protein is an archaeal histone-like protein derived from one of the genus Thermococcus or Pyrococcus; and wherein the transposition system comprises a transposase and an adaptor; The transposition system comprises: (i) TnAa; or (ii) a hyperactive Tn5 transposase and a Tn5-type transposase recognition site; and Wherein the transposition system further comprises one or more oligonucleotides.

13. A method for fragmenting and labeling double-stranded DNA, the method comprising: (a) obtaining a sample containing double-stranded DNA; (b) introducing a fragmentation composition comprising a histone-like protein and a transposition system to the obtained sample to provide a fragmentation reaction mixture; (c) heating the fragmentation reaction mixture at a predetermined temperature for a predetermined amount of time; and (d) isolating the fragmented and tagged DNA from the fragmentation reaction mixture.

14. A method for processing a sample comprising genomic material, the method comprising: (a) obtaining a fragmentation and tagging reaction mixture comprising a transposition system; (b) introducing double-stranded DNA and a histone-like protein into the fragmentation and tagging reaction mixture to provide a fragmentation reaction mixture; and (c) heating the fragmentation reaction mixture to a predetermined temperature for a predetermined amount of time.

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

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

17. A double-stranded DNA fragment having a size ranging between about 250 bp to about 300 bp, wherein the double-stranded DNA fragment is prepared by: (a) obtaining a sample containing double-stranded DNA; (b) introducing a fragmentation composition comprising a histone-like protein and a transposition system to the sample to provide a fragmentation reaction mixture; and (c) heating the fragmentation reaction mixture at a predetermined temperature for a predetermined amount of time.

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