Method and system for library preparation with unique molecular identifiers

Patent metadata

Field	Value
Publication number	US-2018362967-A1
Application number	US-201816013858-A
Country	US
Kind code	A1
Filing date	Jun 20, 2018
Priority date	Jun 20, 2017
Publication date	Dec 20, 2018
Grant date	—

Abstract

Embodiments of a method 100 and/or system 200 or library preparation for sequencing associated with microorganisms can include: preparing a set of unique molecular identifier (UMI)-based molecules associated with one or more targets; preparing a set of sequencing-based primers; generating a set of tagged target molecules based on the set of UMI-based molecules and one or more samples associated with the one or more targets; and/or generating a set of sequencing-ready tagged target molecules based on the tagged target molecules and the set of sequencing-based primers.

First claim

We claim:

1 . A method for library preparation for next generation sequencing (NGS), the method comprising:
preparing a set of unique molecular identifier (UMI)-based primers associated with a set of nucleic acid targets, wherein each UMI-based primer of the set of UMI-based primers comprises:
a UMI region comprising a set of random “N” bases, wherein each random “N” base is selected from any one of an “A” base, a “G” base, a “T” base, and a “C” base; and
a target-associated region associated with at least one nucleic acid target of the set of nucleic acid targets;
preparing a set of sequencing-based primers, wherein each sequencing-based primer of the set of sequencing-based primers comprises an adapter region associated with the NGS; generating a set of tagged target molecules based on a first amplification process with the set of UMI-based primers and at least one sample associated with the set of nucleic acid targets; and generating a set of NGS-ready tagged target molecules based on a second amplification process with the tagged target molecules and the set of sequencing-based primers.

2 . The method of claim 1 , wherein the each UMI-based primer of the set of UMI-based primers further comprises a linker region without full complementarity to the at least one nucleic acid target associated with the target-associated region.

3 . The method of claim 2 , wherein the linker region comprises a length fewer than 21 bases.

4 . The method of claim 2 , wherein, for each UMI-based primer of the set of UMI-based primers, the linker region is positioned between the UMI region and the target-associated region.

5 . The method of claim 2 ,
wherein each UMI-based primer of the set of UMI-based primers further comprises an external adapter region associated with the NGS, wherein the set of tagged target molecules comprises the external adapter regions, and wherein generating the set of NGS-ready tagged target molecules comprises annealing the set of sequencing-based primers with the tagged target molecules at the external adapter regions of the tagged target molecules.

6 . The method of claim 1 , wherein generating the set of tagged target molecules comprises performing the first amplification process with the set of UMI-based primers, the at least one biological sample, and a set of tagging facilitation molecules comprising at least one of MgCl 2 , dimethyl sulfoxide (DMSO), a thermostable nucleic acid binding protein, betaine, formamide, tween, triton, NP-40, Tetramethyl ammonium chloride (TMAC), and bovine serum albumin (BSA).

7 . The method of claim 6 , wherein the thermostable nucleic acid binding protein comprises a thermostable single-stranded DNA binding protein, and wherein generating the set of tagged target molecules comprises performing the first amplification process with the set of UMI-based proteins, the at least one sample, and the set of tagging facilitation molecules comprising MgCl 2 and the thermostable single-stranded DNA binding protein.

8 . The method of claim 1 , wherein generating the set of tagged target molecules comprises performing a purification process with products of the first amplification process to remove UMI-based primers of the set of UMI-based primers from the products of the first amplification process.

9 . The method of claim 1 ,
wherein the first amplification process comprises a first polymerase chain reaction (PCR) process, wherein the second amplification process comprises a second PCR process, wherein the each sequencing-based primer of the set of sequencing-based primers further comprises an index region configured to facilitate multiplexing associated with the NGS; and wherein generating the set of NGS-ready tagged target molecules comprises adding the index region and the adapter region to tagged target molecules of the set of tagged target molecules, based on the second PCR process with the tagged target molecules and the set of sequencing-based primers.

10 . A method for library preparation for next generation sequencing (NGS) sequencing, the method comprising:
generating a set of target-associated amplicons based on a first amplification process with a set of amplicon-generation primers and a set of nucleic acid targets from at least one sample; generating a set of metagenome-associated fragments, based on processing a set of total nucleic acids from the at least one sample; generating a set of sequencing-ready target molecules based on the set of target-associated amplicons, the set of metagenome-associated fragments, and a set of sequencing-based primers, wherein the set of sequencing-ready target molecules is associated with the set of nucleic acid targets.

11 . A method of claim 10 , wherein the set of amplicon-generation primers comprises:
a first subset of amplicon-generation primers, each amplicon-generation primer of the first subset comprising a first amplicon-associated adapter region and a first target-associated region associated with a forward sequence of at least one nucleic acid target of the set of nucleic acid targets; and a second subset of amplicon-generation primers, each amplicon-generation primer of the second subset comprising a second amplicon-associated adapter region and a second target-associated region associated with a reverse sequence of the at least one nucleic acid target of the set of nucleic acid targets, wherein generating the set of target-associated amplicons comprises generating the set of target-associated amplicons based on amplification with the first and the second subsets of amplicon-generation primers.

12 . The method of claim 11 ,
wherein the first subset of amplicon-generation primers comprises first unique molecular identifier (UMI)-based primers, each UMI-based primer of the first UMI-based primers comprising the first amplicon-associated adapter region, the first target-associated region, and a first UMI region; wherein the second subset of amplicon-generation primers comprises second UMI-based primers, each UMI-based primer of the second UMI-based primers comprising the second amplicon-associated adapter region, the second target-associated region, and a second UMI region.

13 . The method of claim 11 , wherein generating the set of metagenome-associated fragments comprises generating the set of metagenome-associated fragments comprising added adapters, based on at least one of a ligation process and an amplification process.

14 . The method of claim 13 , wherein the set of sequencing-based primers comprises:
metagenome-associated adapter regions associated with the NGS and the added adapters of the set of metagenome-associated fragments.

15 . The method of claim 14 , wherein each of the set of sequencing-based primers comprises:
index regions configured to facilitate multiplexing associated with the NGS; and adapter regions associated with the NGS, the set of target-associated amplicons, and the set of metagenome-associated fragments.

16 . The method of claim 15 , wherein the adapter regions of the set of sequencing-based primers are associated with the NGS, the added adapters of the set of metagenome-associated fragments, the first amplicon-associated adapter regions of the first subset of amplicon-generation primers, and the second amplicon-associated adapter regions of the second subset of amplicon-generation primers.

17 . The method of claim 10 , wherein generating the set of metagenome-associated fragments comprises:
generating fragments based on processing the set of total nucleic acids with at least one of an enzymatic

Assignees

• Ubiome Inc

Inventors

• Apte Zachary
• Richman Jessica
• Almonacid Daniel
• Jimenez Juan
• Ortiz Rodrigo
• Morales Eduardo
• Covarrubias Paulo
• Olivares Eduardo
• Ordenes Nicolas
• Leon Luis

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title

• C12Q2525/161

  incorporating target specific and non-target specific sites · CPC title