Transposase-random priming DNA sample preparation
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.
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 .