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EP-3464575-B1 - TRANSPOSASE-RANDOM PRIMING DNA SAMPLE PREPARATION

EP3464575B1EP 3464575 B1EP3464575 B1EP 3464575B1EP-3464575-B1

Inventors

  • AREZI, BAHRAM
  • BORNS, MICHAEL
  • HOGREFE, HOLLY
  • HANSEN, CONNIE

Dates

Publication Date
20260506
Application Date
20170418

Claims (15)

  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, preferably between 2 and 30 linear amplification reaction(s) with a linear amplification primer comprising the first primer sequence at its 3' end.
  3. The method of any prior claim, 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.
  4. The method of claim 3, wherein the first and second sequencing primer sequences are for next generation sequencing applications.
  5. The method of claim 3, 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.
  6. The method of claim 5, wherein the barcode in the adapter and/or random primer is a sample-specific barcode.
  7. The method of claim 5, wherein the barcode in the adapter and/or random primer comprises a degenerate base region (DBR).
  8. The method of any of claims 3-7, 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.
  9. The method of any prior claim, wherein the sample of DNA fragments is isolated from a clinical sample.
  10. The method of claim 9, wherein the clinical sample is cell-free DNA extracted from bodily fluid, preferably from blood.
  11. The method of claim 9, wherein the clinical sample is a formalin-fixed and paraffin embedded (FFPE) sample.
  12. A method comprising: (a) contacting a sample comprising double stranded DNA fragments having an average length of less than 1kb 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 one 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.
  13. The method of claim 12, 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.
  14. The method of any of claims 12-13, 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.
  15. Use of a kit for performing the method of any one of claims 1-14 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 according to claim 1, 3 and 5-7, or a tail primer comprising a 3' sequence that hybridizes to an oligodN tail and a 5' sequence comprising the second primer sequence according to claim 12; 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.

Description

BACKGROUND Next-generation Sequencing (NGS) technologies have made whole-genome sequencing (WGS) routine, and various target enrichment methods have enabled researchers to focus sequencing power on the most important regions of interest. However, there is still a need for better methods of applying NGS to difficult target DNAs, such as formalin-fixed, paraffin-embedded (FFPE) solid tumor samples, cell-free or circulating tumor DNA (cfDNA/ctDNA), or damaged DNA samples. Problems associated with sequencing such targets include that the amounts of DNA can be very small, the DNA can be very short (e.g., fragmented DNA) or chemically modified, and the allele frequencies can be very low. These problems necessitate the capture of many DNA templates. WO 2016/033251 A2, Nugen Technologies Inc., 2016-03-03 and US 2010/120098 A1, Grunenwald Haiying Li et al., 2010-05-13 describes methods, compositions and kits for targeted nucleic acid sequence enrichment in a nucleic acid sample and for high efficiency nucleic acid library generation for next generation sequencing (NGS), where tagmentation with tagged oligonucleotides is performed in one step. The methods only works on long, intact, undegraded DNA strands. In particular, it is challenging to sequence smaller fragments of genomic DNA using transposase-based tagging methods because many of those methods require the insertion of two adjacent transposases to produce a fragment having the appropriate sequencing primer binding site on each end, and such small fragments are often not long enough to accommodate adjacent transposases. The present disclosure provides inter alia compositions and methods to improve tagging and amplifying such DNA samples for downstream analysis, e.g., next generation sequencing. SUMMARY The present invention is defined in the appended claims. The present invention concerns 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. The present invention further concerns a method comprising: (a) contacting a sample comprising double stranded DNA fragments having an average length of less than 1kb 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 one 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. The invention further concerns the use of a kit as defined in the claims. Disclosed herein are methods for tagging DNA fragments in a sample that include: (a) contacting a sample comprising double stranded DNA fragments 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' nucleotide sequence and a 5' nucleotide sequence comprising a second primer sequence; and (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. The PCR ampl