Blocking oligonucleotides for the selective depletion of non-desirable fragments from amplified libraries
AssigneeIllumina, Inc.
InventorsColin Brown, Sarah Shultzaberger, Stephen M. Gross, Angelica Barr, Samantha Snow
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Abstract
The disclosure relates to methods, compositions, and kits for the selective depletion of non-desirable fragments from amplified libraries using blocking oligonucleotides.
Claims41
A method to selectively deplete non-desirable fragments from amplified DNA or cDNA libraries by using one or more blocking oligonucleotides, comprising:
- amplifying in a polymerase chain reaction (PCR), a plurality of library fragments comprising a double stranded template sequence including adapter sequences, wherein a portion of the fragments comprise non-desirable fragments that are not to be analyzed;
- wherein the PCR reaction comprises a plurality of fragments, a polymerase, dNTPS, PCR primers, and one or more blocking oligonucleotides, wherein the one or more blocking oligonucleotides comprise (i) and/or (ii), and (iii):
- (i) at the 5′ terminus, one or more nucleotides that comprise a phosphorothioate linkage;
- and/or
- (ii) at the 3′terminus, one or more nucleotides that comprise a phosphorothioate linkage;
- and
- (iii) a 3′-block that prevent polymerase extension on the 3′ terminus of the blocking oligonucleotide;
- wherein the one or more blocking primers bind to the template sequences of non-desired fragments, thereby blocking amplification of the non-desired fragments by PCR.
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A method to selectively deplete non-desirable fragments from amplified DNA or cDNA libraries by using one or more blocking oligonucleotides, comprising:
- amplifying in a polymerase chain reaction (PCR) reaction, a plurality of library fragments comprising a double stranded template sequence including adapter sequences, wherein a portion of the fragments comprise non-desirable fragments that contain template sequences that are not to be analyzed;
- wherein the PCR reaction comprises a plurality of fragments, a polymerase, dNTPS, PCR primers, and a pool of blocking oligonucleotides, wherein a portion of the pool of the blocking oligonucleotides bind to each strand of a template sequence of a non-desired fragment;
- wherein the one or more blocking primers bind to the template sequences of non-desired fragments, thereby blocking amplification of the non-desired fragments by PCR.
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An RNA-Seq based library preparation kit comprising one or more blocking oligonucleotides, wherein the one or more blocking oligonucleotides comprise (i) and/or (ii), and (iii):
- (i) at the 5′ terminus, one or more nucleotides that comprise a phosphorothioate linkage;
- and/or
- (ii) at the 3′terminus, one or more nucleotides that comprise a phosphorothioate linkage;
- and
- (iii) a 3′-block that prevent polymerase extension on the 3′ terminus of the blocking oligonucleotide;
- wherein the one or more blocking oligonucleotides bind to template sequences of non-desired library fragments, thereby blocking amplification of the non-desired library fragments by PCR.
An RNA-Seq based library preparation kit comprising a pool of blocking oligonucleotides, wherein a portion of the pool of blocking oligonucleotides bind to each strand of a template sequence of a non-desired fragment in a nonoverlapping and adjacent manner, thereby blocking amplification of the non-desired library fragments by PCR.
Dependencies are inferred from the claim text.