Flow Sorting Enrichment and Nanopore Sequencing of Chromosome 1 From a Chinese Individual

Abstract

Sorting of individual chromosomes by Flow Cytometry (flow-sorting) is an enrichment method to potentially simplify genome assembly by isolating chromosomes from the context of the genome. We have recently developed a workflow to sequence native, unamplified DNA and applied it to the smallest human chromosome, the Y chromosome. Here, we modify improve upon that workflow to increase DNA recovery from chromosome sorting as well as sequencing yield. We apply it to sequence and assemble the largest human chromosome - chromosome 1 - of a Chinese individual using a single Oxford Nanopore MinION flow cell. We generate a selective and highly continuous assembly whose continuity reaches into the order of magnitude of the human reference GRCh38. We then use this assembly to call candidate structural variants against the reference and find 685 putative novel SV candidates. We propose this workflow as a potential solution to assemble structurally complex chromosomes, or the study of very large plant or animal genomes that might challenge traditional assembly strategies.

Keywords: chromosome enrichment; chromosome sequencing; chromosome sorting; flow karyotyping; genome assembly; nanopore sequencing; structural variation.

Figure 1

Fold coverage per chromosome. The sequencing is selective for chromosome 1, with all other chromosomes being depleted from the data.

Figure 2

Dot-plot of HG00542 assembly versus GRCh38 chromosome 1. The chromosomes are laid out on the respective axis and a dot denotes aligned sequence between the two assemblies. Bars at the height of 250 Mb on the Y axis show the positions of segmental duplications in GRCh38. The assembly is largely colinear to the reference. The large black block in the center of the dot-plot delimits the 18 Mb centromere of chromosome 1.

Figure 3

Comparative repeat landscapes of GRCh38 chromosome 1 and HG00542 chromosome 1. We find equal resolution across most repeat classes.

Figure 4

Size distribution of SV calls from both Assemblytics and Sniffles at different resolutions. Both call sets have clear peaks around 300 bp corresponding to Alu-elements.

Figure 5

Population frequencies of SV discovered in our assembly that overlap calls by the 1,000 genomes project. We find these SVs to be most frequent in East Asian populations, followed South Asian and American, European, and lastly African populations.