Transposase-random priming DNA sample preparation

Patent metadata

Field	Value
Publication number	US-10240196-B2
Application number	US-201615167823-A
Country	US
Kind code	B2
Filing date	May 27, 2016
Priority date	May 27, 2016
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

Abstract

Provided herein, among other things, are a variety of methods for transposase-mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.

First claim

The invention claimed is:

1. A method comprising:
(a) contacting a sample comprising double stranded DNA fragments having an average length of from 150 bp to 1.5 Kb with a plurality of transposase duplexes each loaded with an adapter to produce adapter-tagged fragments, wherein the adapter comprises a duplex region comprising a transposase recognition sequence and a 5′ overhang region comprising a first primer sequence;
(b) performing a primer extension reaction on the adapter tagged fragments using a random primer to produce random primer extension products, wherein the random primer comprises a random 3′ sequence and a 5′ sequence comprising a second primer sequence;
(c) amplifying the random primer extension products of (b) by polymerase chain reaction (PCR) using a forward primer comprising the first primer sequence at its 3′ end and a reverse primer comprising the second primer sequence at its 3′ end to produce PCR amplification products.

2. The method of claim 1 , wherein prior to step (b) the method further comprises:
(i) performing an extension or extension/ligation reaction on the adapter tagged fragments to fill in the 5′ overhang region and fill in gaps of the adapter tagged fragments; and
(ii) performing at least one linear amplification reaction with a linear amplification primer comprising the first primer sequence at its 3′ end.

3. The method of claim 2 , wherein step (ii) comprises performing between 2 and 30 linear amplification reactions.

4. The method of claim 1 , wherein:
the first primer sequence is a first sequencing primer sequence, the forward primer comprises a 5′ tail comprising a first sequencing primer sequence, or the adapter further comprises a first sequencing primer sequence downstream of the first primer sequence; and
the second primer sequence is a second sequencing primer sequence, the reverse primer comprises a 5′ tail comprising a second sequencing primer sequence, or the random primer further comprises a second sequencing primer sequence downstream of the second primer sequence.

5. The method of claim 4 , wherein the first and second sequencing primer sequences are for next generation sequencing applications.

6. The method of claim 4 , wherein:
the adapter further comprises a barcode downstream of the first sequencing primer sequence; and/or
the random primer further comprises a barcode downstream of the second sequencing primer sequence.

7. The method of claim 6 , wherein the barcode in the adapter and/or random primer is a sample-specific barcode.

8. The method of claim 6 , wherein the barcode in the adapter and/or random primer comprises a degenerate base region (DBR).

9. The method of claim 4 , further comprising sequencing the PCR amplification products to obtain sequence reads for at least a subset of DNA fragments in the sample and assembling the sequence reads into contigs.

10. The method of claim 1 , wherein the sample of DNA fragments is isolated from a clinical sample.

11. The method of claim 10 , wherein the clinical sample is cell-free DNA extracted from a bodily fluid.

12. The method of claim 11 , wherein the bodily fluid is blood.

13. The method of claim 10 , wherein the clinical sample is a formalin-fixed and paraffin embedded (FFPE) sample.

14. A method comprising:
(a) contacting a sample comprising double stranded DNA fragments having an average length of less than 1 kb with a plurality of transposase duplexes each loaded with an adapter to produce adapter-tagged fragments, wherein the adapter comprises a duplex region comprising a transposase recognition sequence and a 5′ overhang region comprising a first primer sequence;
(b) adding an oligo-dN tail to the top strand of the adapter-tagged fragments using an enzyme with terminal transferase activity to produce tailed adapter-tagged fragments;
(c) performing a primer extension reaction on the tailed adapter-tagged fragments using a tail primer to produce tail primer extension products, wherein the tail primer comprises a 3′ sequence that hybridizes to the oligo-dN tail and a 5′ sequence comprising a second primer sequence;
(d) amplifying the tail primer extension products of (c) by polymerase chain reaction (PCR) using a forward primer comprising the first primer sequence at its 3′ end and a reverse primer comprising the second primer sequence at its 3′ end to produce PCR amplification products.

15. The method of claim 14 , further comprising sequencing the PCR amplification products to obtain sequence reads for at least a subset of DNA fragments in the sample and assembling the sequence reads into contigs.

16. The method of claim 14 , wherein the sample of DNA fragments is isolated from a clinical sample, wherein the clinical sample is a formalin-fixed and paraffin embedded (FFPE) sample.

17. A kit comprising:
a plurality of transposase duplexes each loaded with an adapter, wherein the adapter comprises a duplex region comprising a transposase recognition sequence and a 5′ overhang region comprising a first primer sequence;
a random primer comprising a random 3′ sequence and a 5′ sequence comprising a second primer sequence, or a tail primer comprising a 3′ sequence that hybridizes to an oligo-dN tail and a 5′ sequence comprising the second primer sequence;
a forward primer comprising the first primer sequence at its 3′ end;
a reverse primer comprising the second primer sequence at its 3′ end; and
one or more additional reagents for performing the method of claim 1 .

Assignees

• Agilent Technologies Inc

Inventors

• Arezi Bahram
• Borns Michael
• Hogrefe Holly
• Hansen Connie

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12N15/1065

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

• C12N15/1082

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title

• C12Q1/6806 Primary

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