Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations

Abstract

Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods.

Figure 1.

Single-molecule sequencing protocols. The standard single-molecule sequencing protocol using tailed DNA and oligo(dT) flow cells (left) and the novel protocol using Direct Capture (right). The diagram on the left shows three steps in the standard protocol, while the diagram on the right shows the hybridization step only for the Direct Capture protocol with three different possible types of hybridizing molecules: a DNA that is only partially complementary to the capture primer (underhang), a DNA that is perfectly complementary to the capture primer, and a DNA that has a 3′ overhang. All of these molecules are sequenceable.

Figure 2.

Sequence reads from representative region using Direct Capture and JumpStart Sequencing. Representative sequence reads from one region of BRCA1 exon 16 in the forward (black) and reverse (blue) directions are shown for the NA14096 sample. In the top half of the figure with Direct Capture Sequencing, horizontal black and blue arrows show where sequencing begins after each capture primer. In the lower half of the figure with JumpStart Sequencing, start sites were randomized by extension with polymerase prior to sequencing. A histogram for start sites is shown in Supplemental Figure S4. Sequence reads are aligned to the reference in the center (bold) and differences noted in red for substitutions, with dashes for deletions, and with bolded, underlined bases for insertions. Gaps are included between sequences so that individual reads can be distinguished. Random errors occur throughout the reads, but these are easily distinguished from real variants in which differences from the reference occur much more often (downward red arrow). This position corresponds to dbSNP variant rs1799966, a common substitution SNP. The reads shown are a sampling of those obtained for this region.

Figure 3.

Sequence coverage. Coverage for 19 of the DNAs listed in Table 1 that were hybridized and sequenced on the same flow cell is shown for 500 bp of exon 11. Coverage bins of 5 bp were normalized to achieve the same amount of total coverage for each sample by multiplying by the appropriate factor (range = 0.70 to 2.7). DNA sample NA13707, which contains a heterozygous 40-bp deletion in this region (heavy black line); all others (thin gray lines). Numbering for the region is as found in the BIC database. The lowered coverage over a larger region than 40 bp and not centered on the deletion is caused by the presence of primers located entirely within or overlapping the deletion that generate no reads from those primers for the deletion allele.

Figure 4.

Deletion sequences. The exact coordinates of the 40-bp deletion in NA13707 can be determined by examining reads that map to the reference sequence in the region of interest in an interrupted manner. The reference sequence is shown in the center with the known 40-bp deletion region underlined. From 928 reads aligning to this region, 28 selected reads with different end points are shown. Forward reads (left to right) are in black, and reverse reads (right to left) are in blue. Deletions relative to the reference are shown by dashes and insertions by bolding and underlining the adjacent base.