Abstract: The disclosure provides DNA library preparation methods that do not require a purification between adapter ligation and PCR amplification. Adaptors are added to DNA fragments to form oligonucleotide extension products and the oligonucleotide extension products are amplified without stopping or interruption for a cleanup step. Excess materials, such as enzymes, adaptors, or co-factors, from the adaptor addition step do not interfere with the amplification step and the amplification step proceeds without regards to the presence of reagents from the ligation step. In preferred embodiments, the ligation and amplification step make use of a common priming sequence e.g., in the form of one of the adaptor oligos.

Abstract: Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).

Abstract: Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).

Abstract: Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).

Abstract: The disclosure provides DNA library preparation methods that do not require a purification between adapter ligation and PCR amplification. Adaptors are added to DNA fragments to form oligonucleotide extension products and the oligonucleotide extension products are amplified without stopping or interruption for a cleanup step. Excess materials, such as enzymes, adaptors, or co-factors, from the adaptor addition step do not interfere with the amplification step and the amplification step proceeds without regards to the presence of reagents from the ligation step. In preferred embodiments, the ligation and amplification step make use of a common priming sequence e.g., in the form of one of the adaptor oligos.

Abstract: Provided herein are compositions, systems, and methods employing hyper-thermostable lysine-mutant ssDNA/RNA ligases that possesses both ssRNA ligase and ssDNA ligase activity. In certain embodiments, such hyper-thermostable lysine-mutant ssDNA/RNA ligases are used to ligate an first single stranded nucleic acid sequence with a 5? adenylated end to a second single stranded nucleic acid sequence (e.g., at a temperature of at least 75° C.) to form a ligated nucleic acid sequence. In further embodiments, the ligated nucleic acid sequence is sequenced.

Abstract: Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).