Linear assembly of a human centromere on the Y chromosome

Abstract

The human genome reference sequence remains incomplete owing to the challenge of assembling long tracts of near-identical tandem repeats in centromeres. We implemented a nanopore sequencing strategy to generate high-quality reads that span hundreds of kilobases of highly repetitive DNA in a human Y chromosome centromere. Combining these data with short-read variant validation, we assembled and characterized the centromeric region of a human Y chromosome.

Figure 1. BAC-based longboard nanopore sequencing strategy on the MinION.

(a) Optimized strategy to cut each circular BAC once with transposase results in a linear and complete DNA fragment of the BAC for nanopore sequencing. (b) Yield plot of BAC DNA (RP11-648J18). (c) High-quality BAC consensus sequences were generated by multiple alignment of 60 full-length 1D reads (shown as blue and yellow for both orientations), sampled at random with ten iterations, followed by polishing steps (green) with the entire nanopore long-read data and Illumina data. (d) Circos representation of the polished RP11-718M18 BAC consensus sequence. Blue arrowheads indicate the position and orientation of HORs. Purple tiles in yellow background mark the position of the Illumina-validated variants. Additional purple highlight extending from select Illumina-validated variants are used to identify single-nucleotide-sequence variants and mark the site of the DYZ3 repeat structural variants (6 kb) in tandem.

Figure 2. Linear assembly of the RP11 Y centromere.

(a) Ordering of nine DYZ3-containing BACs spanning from proximal p-arm to proximal q-arm. The majority of the centromeric locus is defined by the DYZ3 conical 5.8-kb HOR (light blue). Highly divergent monomeric alpha satellite is indicated in dark blue. HOR variants (6.0 kb) indicated in purple. (b) The genomic location of the functional Y centromere is defined by the enrichment of centromere protein A (CENP-A), where enrichment (∼5–6×) is attributed predominantly to the DYZ3 HOR array.