TECHNICAL FIELD

The invention relates to the preparation of DNA libraries for next-generation sequencing.

BACKGROUND

Next-generation sequencing (NGS) of DNA can rapidly provide large amounts of medically important genetic information. NGS sequencing instruments operate on libraries of DNA that are prepared from clinical or biological samples. DNA library preparation often uses a commercial kit that comes with the reagents and instructions for the isolation of genes of interest. One commonly used library prep kit is the library prep kit sold under the trademark TRUSEQ by Illumina (San Diego, Calif.).

Typical library preparation kits and protocols provide for the fragmenting of input DNA followed by end repair, bead cleanup, A-tailing, adaptor ligation, bead cleanup, PCR amplification, and a final bead cleanup. The cleanup steps may proceed by other known methods, but cleanup using magnetic beads is popular as it is reasonably straightforward using commercially available kits and instructions from companies such as Agencourt.

While time consuming, bead cleanup is understood to be a necessary step in library preparation because excess reagents from one step, if not removed, will interfere with or prevent successful completion of subsequent steps. Specifically, purification is understood to be required after ligation to remove adaptors and other ligation reaction components such as high concentrations of magnesium and PEG, which are incompatible with the subsequent PCR amplification. Furthermore, existing protocols require distinct PCR primers in order to amplify a functional final sequencing library.

SUMMARY

The disclosure provides DNA library preparation methods that do not require a purification between adapter ligation and PCR amplification and in which adapter oligos can function as primers during amplification. In preferred embodiments, adaptors are added to DNA fragments and then the fragments are amplified without an intervening cleanup step. In fact, at least one oligo strand of the starting adaptors may, in some embodiments, function as an amplification primer.

In some embodiments, partially double-stranded adaptors are ligated at both ends of a DNA fragment, to the free 5′ ends, after which the 3′ end is extended to copy the full adapter sequence. The adaptor may be at least partially double-stranded to facilitate recognition and enzymatic by a ligase. After the fragments are extended across the major strands of the adaptors, displacing the minor strands of the adaptors, the resultant adaptor-ligated fragments are amplified by polymerase chain reaction (PCR). The disclosure includes results showing that no purification or cleanup step is required between adaptor ligation and amplification. Thus materials present for adaptor ligation may still be present during amplification including materials such as the adaptors, enzymes, co-factors, etc. Other embodiments of adaptors and adaptor addition are within the scope of the disclosure.

In certain embodiments, a first adaptor is ligated to a DNA fragment and a second adaptor hybridizes thereto, after which the second adaptor is extended through the first to form an oligonucleotide extension product which is amplified. The amplification can even use the second adaptor as a primer. The oligonucleotide extension products are amplified without stopping or interruption for a cleanup step. Excess materials, such as enzymes, adaptors, or co-factors, from the adaptor ligation step do not interfere with the amplification step and the amplification step proceeds without regards to the presence of reagents from the ligation step. In fact, in preferred embodiments, the ligation and amplification step make use of a common primer, the second adaptor oligo.

Methods of the disclosure are useful with single-primer enrichment in which target-specific primers and adaptors are used in the adaptor ligation steps and then are also used as primers in the amplification step. In such embodiments, target DNA is fragmented, and indexed forward adapters are ligated. Oligos comprising target probes and reverse adaptors are annealed to the fragments and extended. The resulting ds product is denatured and PCR primers are annealed for amplification and library enrichment.

Embodiments of the methods need no more than a single purification or bead cleanup after the PCR step. Methods of the disclosure are compatible with both mechanical and enzymatic shearing of DNA. Adaptors used according to the disclosure allow for both ligation and PCR amplification, without addition of distinct PCR primers (which are required with Illumina Y-adaptors). Thus, the disclosure provides library preparation methods in which a post-ligation bead cleanup is eliminated. Library preparation according to methods of the disclosure may proceed with no more than a single purification or bead cleanup step and generate high quality libraries for sequencing. The library preparation methods use sequencing adaptors that also serve as PCR primers. Additionally, methods of the disclosure are compatible with both enzymatic and mechanical DNA fragmentation.

Aspects of the invention provide a method of preparing a sequencing library. The method includes obtaining a plurality of DNA fragments from nucleic acid from a sample, incubating the DNA fragments with adaptor oligos to form adaptor-ligated fragments in which at least a first adaptor oligo is ligated to a fragment and at least a second adaptor oligo hybridizes to the fragment and is extended by a polymerase to form a sequence complementary to a target and complementary to the first adaptor oligo, and amplifying the DNA fragments in the presence of the adaptor oligos to form a plurality of amplicons. Copies of the second adaptor oligo function as primers during the amplification step.

In certain embodiments, the adaptor ligation and amplification includes (a) appending a first adaptor to a 5′ end of each DNA fragment; (b) annealing one or more second adaptor oligos to the DNA fragments, whereby each of the one or more oligonucleotides comprise a 3′ portion that is complementary to a sequence of interest present in one or more of the fragments, and a 5′ portion comprising a second adapter sequence; (c) extending the one or more second adaptor oligos with a polymerase thereby generating one or more oligonucleotide extension products with the first adaptor at a first end and the second adaptor sequence at a second end; and (d) amplifying—with no intervening cleanup or purification step—the one or more oligonucleotide extension product using the second adaptor oligos as an amplification primer, to enrich for nucleic acid fragments containing the first adaptor and the second adaptor sequence at each end.

Methods optionally include purifying the amplicons to remove excess material. The amplicons may be attached to a flow cell surface to form sequencing clusters. Methods may include sequencing the amplicons to determine a sequence of the nucleic acid. In some embodiments, the methods begin with any of reverse transcribing RNA to obtain DNA nucleic acid and/or fragmenting the nucleic acid from the sample to obtain the plurality of DNA fragments.

