Completely phased genome sequencing through chromosome sorting

Abstract

The two haploid genome sequences that a person inherits from the two parents represent the most fundamentally useful type of genetic information for the study of heritable diseases and the development of personalized medicine. Because of the difficulty in obtaining long-range phase information, current sequencing methods are unable to provide this information. Here, we introduce and show feasibility of a scalable approach capable of generating genomic sequences completely phased across the entire chromosome.

Fig. 1.

Schematic diagram of Phase-Seq work flow. Single chromosomes were sorted into wells of a 96-well plate in which single chromosome amplifications were performed. Each amplified DNA molecule from a single chromosome (e.g., Chr19) contained a specific tag (shown in red or blue) that allowed multiplex sequencing on a high-throughput sequencing platform. Multiplexed reads were assigned to haploid genomes based on the combination of single chromosome-specific tags (shown in red or blue) and haploid genome-specific SNPs (lowercase letters in italic bold type). (Inset) FACS sorting of stained single chromosomes is based on the fluorescence patterns of Hoechst and Chromomycin, which allow reliable separation of different chromosomes (Chr18 and Chr19 are marked).

Fig. 2.

Sequence reads from sorted and amplified single chromosomes predominantly map to Chr19. (A) The count (in 10⁵) of aligned reads that map to each chromosome. (B) The average mapping quality score for each chromosome. For both A and B, the color codes for the value of the next_phred mapping quality score cutoff (co) used to retain the reads. (C) The percentage of reads that map to Chr19 at various quality score cutoffs (0, 20, 40, 60, 80, and 100).

Fig. 3.

Distribution of reads along Chr19. (A) The full span of Chr19 is divided into about 600 nonoverlapping windows of size 10⁵ base each, and the counts of high-quality reads (next_phred mapping quality ≥100) for each window are displayed as color-coded data points, indicating the percentage of bases in each window being covered by the reads for at least one (red), two (green), five (purple), and twenty (blue) times. (B) Plot of total size of all gaps exceeding a certain size threshold. Details are given in Inset (e.g., there are 18 gaps larger than 10 Kb, and in total, they covered 475 Kb of Chr19).