High-Fidelity Nanopore Sequencing of Ultra-Short DNA Targets

Abstract

Nanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultrashort DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNVs) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. After sequencing on the Oxford Nanopore Technologies MinION platform, the resulting repeat sequences can be aligned to produce a highly accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences <100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of <10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resource-limited settings.

Figure 1

Sequencing ultrashort reads on the MinION. (a) (1) Molecular inversion probes (MIPs) anneal adjacent to the target sequence (blue) at anchor site 1 (AS1, orange) and anchor site 2 (AS2, green). Phusion polymerase copies the target sequence into the MIP; the lack of 5′ → 3′ exonuclease activity ensures that extension halts when the polymerase reaches AS2. (2) Ampligase ligates the extended template to the phosphorylated 5′ end of the MIP, generating circular ssDNA. Linear ss- or dsDNA fragments are degraded by a combination of exonuclease I and exonuclease III. (3) The circular DNA is subjected to RCA to generate tandem repeats of the original target, yielding ultralong, concatemerized ssDNA. (4) The RCA product is converted to dsDNA with Taq polymerase and subjected to ONT library preparation. (5) Sequencing reads are collected from a new MinION R9.4 flow-cell run for 24 h. (b) The raw sequences are compiled and analyzed. The identified repeats have poor accuracy in isolation, but since the sequencing errors vary across repeats, they can be aligned together to produce a high-fidelity consensus sequence.

Figure 2

Circularization can be performed on target sequences as short as a single nucleotide with reaction efficiency that is independent of target sequence length. After five rounds of temperature cycling and subsequent exonuclease treatment, we achieve consistently efficient circularization for target sequences ranging in length from 1 to 120 nt (lanes 2–8). In this denaturing gel, lane 1 contains a mixture of all the linear ssDNA target sequences. The lengths listed are the lengths of the target region; the full lengths in lane 1 have additional flanking 28- and 23-nt anchor sites, and the full lengths in lanes 2–8 have an additional 102 nt from the MIP. Lane 9 illustrates that no circular DNA is produced in the absence of the target sequence.

Figure 3

Improving read accuracy through repeat-based consensus. (a) Representative consensus sequence generation. A single, base-called read is split into its individual repeats. These repeats are aligned with each other to generate a consensus sequence via a winner-take-all base-calling strategy. Gaps are removed and the consensus sequence is then compared back to the original sequence to assess the postalignment accuracy. (b) Histogram of alignment scores before (gray) and after (red) consensus sequence generation. The “before” alignment score is an average over the alignment scores of all the repeats found within a single raw read. Data includes all reads with more than three identified repeats, regardless of the quality score or pass/fail designation of the MinION software.

Figure 4

Increased accuracy from alignment of tandem repeats. Plots show normalized Smith-Waterman alignment scores as a function of the number of repeats before (gray) and after (red) alignment. Before consensus sequence generation, alignment score exhibits no dependence on repeat count. Since each “before point” represents an average over all repeats in that read, the observed narrowing arises solely because the increased number of repeats decreases the standard deviation of the average alignment score. After the consensus sequence is generated, the alignment accuracy exhibits a strong dependence on the number of repeats used.

Figure 5

Quantitative analysis with HiFRe. a) Counting relative molecular abundance for two sequences present in mixtures at different ratios. A linear fit to y = mx + b yielded m = 0.97 ± 0.04 and b = 0.02 ± 0.02 with R² = 0.993. This strong linear relationship results in a limit of detection of 3.3 ± 2.1%. b) Discrimination and quantitation of SNVs in short DNA sequences. Two sequences differing by three SNVs were mixed together in different ratios, and the plot shows the output ratios recovered after HiFRe analysis. Green bars represent the fraction of the original sequence and yellow bars show the sequence with three SNVs. In both panels, the error bars represent the standard deviation for the mean of two multiplexed sequencing runs.