CROSS-REFERENCING

This application is a § 371 national phase of International Application No. PCT/IB2016/055251, filed on Sep. 1, 2016, which claims the benefit of UK patent application serial no. 1515557.5, filed on Sep. 2, 2015, which applications are incorporated by reference herein.

BACKGROUND

Many diseases are caused by somatic mutations. Because somatic mutations only occur in a fraction of the cells in the body, they can be very difficult to detect by next generation sequencing (NGS). One problem is that every library preparation method and sequencing platform results in sequence reads that contain errors, e.g., PCR errors and sequencing errors. While it is sometimes possible to correct systematic errors (e.g., those that are correlated with known parameters including sequencing cycle-number, strand, sequence-context and base substitution probabilities), it is often impossible to figure out with any certainty whether a variation in a sequence is caused by an error or if it is a “real” mutation. This problem is exacerbated if the amount of sample is limited and mutation-containing polynucleotides are present only at relatively low levels, e.g., less than 5%, in the sample. For example, if a sample contains only one copy of a mutation-containing polynucleotide in a background of hundreds of polynucleotides that are otherwise identical to the mutation-containing polynucleotide except that they do not contain the mutation, then, after those polynucleotides have been sequenced, it is often impossible to tell whether the variation (which may only be observed in about 1/100 of the sequence reads) is an error that occurred during amplification or sequencing. Thus, the detection of somatic mutations that cause diseases can be extremely difficult to detect with any certainty.

Schmitt et al (Proc. Natl. Acad. Sci. 2012 109: 14508-14513) proposed a solution that involves tagging a sample with custom Y-shaped adaptors. The Y-shaped adaptors were generated by first introducing a single-stranded randomized nucleotide sequence into the stem region of one adaptor strand. A second adaptor strand, is then extended using a DNA polymerase, to generate a Y-shaped adaptor where the stem region has a complementary double-stranded randomized nucleotide sequence. Adaptors are then tailed by adding a 3′ base overhang using a DNA polymerase. The overhang assists in adaptor ligation to fragmented DNA tailed with a complementary base. Adaptors are ligated to fragmented DNA before the library is PCR amplified, using primers that hybridize to the single stranded (non-stem) region of the adaptors. Tag sequences allow reads deriving from the top strands of fragmented DNA to be discriminated from sequence reads derived from bottom strands. This requires paired-end sequencing and comparison of both read 1 and read 2 tag sequences. Schmitt's method is based on the idea that for a true DNA mutation, complementary substitutions should be present on both strands and, as such, a mutation can only be called with confidence if it is present in sequences from both strands.

While useful for discriminating between sequence reads from top and bottom strands, Schmitt's method has several limitations.

First, because sequence tags are random it can be difficult to identify tags that have been ‘mutated’ due to PCR or sequencing errors. In addition, it is difficult to detect errors that occurred during oligonucleotide synthesis, such as n-1 deletions.

Second, manufacture of the double-stranded adaptors is complex and expensive. Typically, Y-shaped adaptors are manufactured by annealing two oligonucleotides. In contrast, Schmitt's method also includes incorporation of random bases into the oligonucleotide, DNA polymerase extension and tailing different 3′ bases onto adaptors and fragmented DNA. These steps can be inefficient and require additional purification and quality control checks.

Third, it is difficult to control the relative incorporation of different bases in the degenerate tag sequence during oligonucleotide synthesis. This can result in some tag sequences being present at higher levels than others in the pool of Y-shaped adaptors, which reduces the probability that a fragment is tagged with unique tag sequences.

Fourth, because the tags are attached to the template in bulk, a number of tag sequences are required to reduce the probability of different fragments being attached to adaptors containing the same tag sequences and to improve the chance of detecting a PCR and/or sequencing error that results in one tag being ‘mutated’ into another. As a result, the tag sequences are typically relatively long; for example, Schmitt use 12 nucleotide tag sequences. However, long runs of random bases are likely to form intra- and inter-molecular hybrids, which can cause problems for downstream applications such as in-solution hybridization. In this application, adaptor and index sequences are ‘masked’ to reduce the effect of inter-molecular hybridisation by including blocking oligonucleotides in the hybridization. However, masking the degenerate region of the tag requires incorporation of ‘universal’ bases such as inosine, with associated additional costs. In addition, tag sequences use up a proportion of each sequencing read thereby reducing the sequence data from target fragment(s). This effect is increased if tag sequences are longer.

Fifth, in the Schmitt protocol the PCR is performed on a pool of molecules, each tagged with different 5′ and 3′ random tag sequences. Failure to remove residual adaptors can result in hybridization of an adaptor strand to a template molecule during PCR, which can inadvertently tag a molecule with a different tag sequence.

Finally, Schmitt tag DNA fragments with both 5′ and 3′ random tag sequences. If, by chance, the 5′ and 3′ tag sequences are complementary, or partially complementary, then the tags can intra-molecularly hybridize resulting in suppression of PCR amplification. This can result in uneven amplification of template molecules, depending on their 5′ and 3′ tag combination.

The present disclosure provides an alternative, better, way for tagging DNA molecules in a way that the sequence reads from top and bottom strands can be discriminated.

SUMMARY

Described herein is a method of sequencing. In some embodiments the method may comprise: splitting an asymmetrically tagged library into a plurality of subsamples, tagging the adaptor-ligated DNA in the sub-samples with sequence tags that identify the sub-samples, optionally pooling the sub-samples, sequencing polynucleotides from each of the tagged sub-samples, or copies of the same, to produce sequence reads each comprising: (i). a sub-sample identifier sequence and (ii). the sequence of at least part of a fragment in the sample, where at least some of the sequence reads are derived from the top strand of a fragment in the sample and some of the sequence reads of are derived from the bottom strand of the same fragment.

As will be described in greater detail below (and illustrated inFIG. 1), the method provides sequence reads in which sequence reads that are derived from different strands (i.e., the top and bottom strands) of the same fragment (i.e., the same original double-stranded molecule) can be distinguished. In addition, the method provides a way to distinguish sequence reads that are derived from fragments that are otherwise identical. These features allow one to identify sequence variation with exceptional confidence.

The method finds particular use in analyzing samples of DNA in which the amount of DNA, or diversity of fragmentation breakpoints, is limited and/or that contain fragments having a low copy number mutation (e.g. a sequence caused by a mutation that is present at low copy number relative to sequences that do not contain the mutation). These features are often present in patient samples that can be obtained non-invasively, e.g., circulating tumor (ctDNA) samples, which can obtained from peripheral blood, or invasively, e.g., tissue sections. In such samples, the mutant sequences may only be present at a very limited copy number (e.g., less than 10, less than 5 copies or even 1 copy in a background of hundreds or thousands of copies of the wild type sequence) and there is a high probability that at least some of the mutant fragments have an otherwise identical sequence (including identical ends) to a wild type fragment. In these situations, it can be almost impossible to identify a sequence variation with significant confidence.

The present method—because it involves splitting, and tagging, the sample after it is tagged with a “generic” asymmetric adaptor—has multiple advantages over the prior methods, e.g., the Schmitt method summarized above. For example, sequence tags used for each sub-sample can be error-correctable rather than random sequences. Therefore errors in the tag sequences can be recovered, retaining many reads that would otherwise be misassigned or rejected by the analysis pipeline. Second, manufacture of adaptors in the present method is more straightforward than the method used by Schmitt. Adaptors can be manufactured by annealing oligonucleotides thereby avoiding enzymatic steps and subsequent purification and quality controls steps. Third, unlike degenerate oligonucleotide synthesis, non-degenerate oligonucleotides do not have synthesis biases. Fourth, because tag sequences can be designed, rather than random, they can be of specified length and sequence composition. For example, this can allow design of specific blocking sequences, tailoring the length of tags to a specific sequencing platform or avoiding secondary structure(s). Fifth, generic adaptors do not include tag sequence(s) therefore residual adaptor strands cannot inadvertently hybridize to a molecule and add a different tag sequence. Sixth, tag combinations can be rationally designed rather than random combinations of different 5′ and 3′ random tag sequences. Therefore tag combinations can be chosen to minimize uneven amplification of template molecules.

