US 11,965,157 B2
Compositions and methods for library construction and sequence analysis
Jeffrey A Gole, San Diego, CA (US); Athurva Gore, San Diego, CA (US); and Rui Liu, Shanghai (CN)
Assigned to Singlera Genomics, Inc., La Jolla, CA (US)
Appl. No. 16/605,203
Filed by Singlera Genomics, Inc., La Jolla, CA (US)
PCT Filed Apr. 18, 2018, PCT No. PCT/US2018/028191
§ 371(c)(1), (2) Date Oct. 14, 2019,
PCT Pub. No. WO2018/195217, PCT Pub. Date Oct. 25, 2018.
Claims priority of provisional application 62/657,544, filed on Apr. 13, 2018.
Claims priority of provisional application 62/487,423, filed on Apr. 19, 2017.
Prior Publication US 2020/0123538 A1, Apr. 23, 2020
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6874 (2018.01)
CPC C12N 15/1093 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6874 (2013.01)] 17 Claims
 
1. A method, comprising ligating a set of adaptors to a library of single-stranded polynucleotides, wherein:
each single-stranded polynucleotide in the library of single-stranded polynucleotides comprises a target sequence;
the ligation is catalyzed by a single-stranded DNA (ssDNA) ligase;
each single-stranded polynucleotide is blocked at the 5′ end to prevent ligation at the 5′ end;
each adaptor comprises a unique molecular identifier (UMI) sequence that earmarks the single-stranded polynucleotide to which the adaptor is ligated, and is blocked at the 3′ end to prevent ligation at the 3′ end; and
the 5′ end of the adaptor is ligated to the 3′ end of the single-stranded polynucleotide by the ssDNA ligase to form a linear ligation product,
thereby obtaining a library of linear, single-stranded ligation products, wherein each linear, single-stranded ligation product comprises a single adaptor;
further comprising converting the library of linear, single-stranded ligation products into a library of linear, double-stranded ligation products;
further comprising amplifying the library of linear, double-stranded ligation products by a polymerase chain reaction (PCR), using a set of primers each comprising a sequence that is reverse-complement to the adaptor and/or hybridizable to the adaptor, and a primer hybridizable to the target sequence, thereby obtaining an amplified library of linear, double-stranded ligation products comprising sequence information of the target sequence; and
further comprising sequencing the amplified library of linear, double-stranded ligation products, wherein the conversion rate of the sequencing (percentage of single-stranded polynucleotides in the library that gives rise to sequencing reads) is at least 60%.