RANDOM ARRAY SEQUENCING OF LOW-COMPLEXITY LIBRARIES

• US 20130065768A1
• Filed: 08/28/2012
• Published: 03/14/2013
• Est. Priority Date: 09/12/2011
• Status: Active Grant

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Sequenta LLC
• Inventors: Zheng, Jianbiao, Willis, Thomas, Faham, Malek
• Granted Patent: US 10,385,475 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: C40B 20/00 Methods specially adapted for identifying library members
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support

Abstract

The invention is directed to a method of sequencing low-complexity amplicons randomly arrayed at high density on a surface. Methods of the invention include preparing amplicons for sequencing by a sets of primers that ensure initial signals front different amplicons on the surface will be evenly distributed among the different nucleotides being added in a sequencing by synthesis operation.

Claims

1. A method of sequencing by synthesis a library of homologous template nucleic acids, the method comprising the steps of:

amplifying the library with a plurality of printers to form an amplicon of homologous templates, the plurality of primers including for each template more than one forward primer and at least one reverse primer, wherein each of the more than one forward primers has a 3′

region complementary to at least one template and a 5′

tail comprising a sequencing primer binding site, and wherein at least one of the more than one forward primers has from one to three nucleotides inserted between the 5′

tail and the 3′

region so that whenever a sequencing primer specific for the sequencing primer binding site anneals thereto and is extended by a first nucleotide, on average a quarter of the forward primers are extended by a different first nucleotide;

randomly arraying nucleic acids of the amplicon of homologous templates on a surface;

amplifying the randomly arrayed nucleic acids to form a random array of template amplicons;

identifying isolated, contiguous and overlapping template amplicons by extending a sequencing primer annealed to the sequencing primer binding site by at least one nucleotide; and

sequencing by synthesis isolated, contiguous and overlapping template amplicons to determine nucleotide sequences of homologous template nucleic acids of the library.

2. The method of claim 1 wherein said more than one forward primers has from one to two wildcard nucleotides inserted between said 5′

tail and said 3′

region.

3. The method of claim 2 wherein said library of homologous templates is a population of recombined immune molecules.

4. The method of claim 3 wherein said recombined immune molecules are nucleic acids encoding T cell receptors or portions thereof or nucleic acids encoding immunoglobulins or portions thereof.

5. The method of claim 4 wherein said recombined immune molecules are at least, fifty percent homologous to one another.

6. The method of claim 1 wherein said more than one forward primers are arranged in groups of up to four primers each, such that the primers of each group have a first nucleotide position, a second nucleotide position, a third nucleotide position and a fourth nucleotide position juxtaposed to and downstream of said sequencing primer binding site, wherein up to one nucleotide in the first nucleotide position is a nucleotide from said 35 region of a first primer of a group, up to two nucleotides in the second nucleotide position are nucleotides from said 3′

regions of the first primer and a second primer of the group, up to three nucleotides in the third nucleotide position are nucleotides from said V regions of the first primer, the second primer and a third primer of the group, and up to four nucleotides in the fourth nucleotide position are nucleotides from said 3′

regions of the first primer, the second primer, the third printer and a fourth primer, and wherein remaining nucleotides in the first, second, third and fourth positions are selected to maximize nucleotide diversity in each of such positions.

7. The method of claims 6 wherein said library of homologous templates is a population of recombined immune molecules.

8. The method of claim 7 wherein said population of recommitted immune molecules is a population of nucleic acids encoding T cell receptors or fragments thereof or a population of nucleic acids encoding B cell receptors or fragments thereof.

9. The method of claim 8 wherein said recombined immune molecules are at least fifty percent homologous to one another.